Panax ramosa rare saponin composition, preparation method and application thereof

Abstract

The present application relates to a rare ginsenoside composition and a method and application thereof, wherein the ginsenoside composition comprises 0.02-0.1 mg / mL of S-Rg3, 0.009-0.029 mg / mL of R-Rg3, 0.087-0.245 mg / mL of Rg5 and 0.013-0.044 mg / mL of CK. The rare ginsenoside is prepared by fermentation, which comprises the following steps: step-by-step fermentation with yeast and lactic acid bacteria. Through the step-by-step fermentation method, the inherent ginsenoside is converted into rare ginsenoside, and 17 kinds of rare ginsenosides, including S-Rg2, R-Rg2, F1, F4, Rk3, F2, Rh4, S-Rg3, R-Rg3, PPT, Rg5, CK, S-Rh2, R-Rh2, Rk2, Rh3 and PPD, are obtained. The bioactivity of the fermented ginseng is higher, and the fermented ginseng can be applied to the food, medicine, health food and skin care product industries.

Description

Technical Field

[0001] This invention relates to the field of ginsenosides, specifically to a method for preparing rare ginsenoside products by stepwise fermentation. Background Technology

[0002] Ginseng is a traditional Chinese herbal medicine, and ginsenosides, as active ingredients, are widely accepted for their well-defined functional effects. Ginsenosides account for 3-4% of ginseng content, and there are many types, with over 70 identified. Ginseng's inherent saponins are triterpenoid compounds, which, according to their structural type, can be divided into tetracyclic triterpenoids (dammarane type), pentacyclic triterpenoids, and oleanolic acid type ginsenosides. Pentacyclic triterpenoids (oleanolic acid type) ginsenosides are present in extremely low amounts in ginseng. Tetracyclic triterpenoids (dammarane type) ginsenosides, based on the different structures at C-3, 6, and 20 positions on the tetracyclic core, can be further divided into protopanaxadiol type and protopanatriol type. According to the different substitution positions of the chiral C (C-20), protopanaxadiol and protopanatriol type ginsenosides are further classified into 20(S) and 20(R) types. Based on the different sugar chains at the C-3 and C-20 positions (mostly glucose, arabinose, and xylose), the protopanaxadiol-type ginsenosides in ginseng mainly include Ra1, Ra2, Ra3, Rb1, Rb2, Rb3, Rc, and Rd. Protopanaxadiol-type ginsenosides, upon hydrolysis, lose the sugar chains at the C-3 and C-20 positions, forming hydrolysis products such as Rg3, F2, Rh2, and CK, with the final aglycone being protopanaxadiol (PPD). Based on the different ligand sugar groups at the C-6 and C-20 positions (mostly glucose, rhamnose, and xylose), protopanatriol-type ginsenosides mainly include Re, Rf, and Rg1. After hydrolysis and loss of the sugar chains at the C-6 and C-20 positions, they form rare saponin intermediates such as Rh1 and F1, with the final aglycone being protopanatriol (PPT).

[0003] Ginsenosides Ra, Rb, Rc, Rd, Re, Rf, and Rg1 are inherent saponins in ginseng, and their content in ginseng is relatively high, accounting for more than 95% of the total saponins. After hydrolysis, inherent ginsenosides lose their coordinating sugar chains to obtain ginsenosides Rh1, Rh2, F2, F1, CK, PPD, and PPT, etc., which are present in very small amounts or almost none in ginseng and are called rare ginsenosides.

[0004] Ginsenosides, inherent to ginseng, have low absorption rates and poor bioactivity in the gastrointestinal tract. Under the acidic conditions of the stomach and the action of intestinal bacteria, they undergo degradation and deglycosylation in the gastrointestinal tract, transforming into rare ginsenosides, which are then absorbed and utilized by the body. Deglycosylated rare ginsenosides are more easily absorbed into the bloodstream and maintain higher bioactivity. Numerous pharmacological studies have shown that rare ginsenosides exhibit unique therapeutic effects in anti-inflammatory, antitumor, and antioxidant aspects. Chen Ling ("The Influence of Lactic Acid Bacteria Fermentation on Ginsenosides and Their Antitumor Activity," Jilin University, 2017) studied the fermentation preparation of ginsenosides and conducted a preliminary study on their in vitro antitumor activity. The fermented sample showed a higher inhibition rate against HepG2 cells than the unfermented sample.

[0005] There are numerous existing patents on the fermentation and transformation of ginsenosides. CN 106498018 A discloses a method using Rb1 or ginseng, American ginseng, and Panax notoginseng as raw materials, employing preferred yeasts, lactic acid bacteria, and Bifidobacteria that produce β-glucosidase, fermenting at 30-37℃ for 5-14 days to conduct a complex microbial transformation, obtaining rare ginsenoside CK. Although this method uses yeast and lactic acid bacteria for fermentation and transformation, the two are fermented simultaneously, and only the transformation effect of rare ginsenoside CK is considered after fermentation. CN1738905A discloses ginseng fermented with lactic acid bacteria, yogurt containing lactic acid bacteria-fermented ginseng, and the lactic acid bacteria used in its preparation. CN103099832 B discloses a method for the biotransformation of Panax notoginseng using Candida albicans. This method uses Candida albicans as the fermentation strain to transform Panax notoginseng saponins, mainly comparing the fermentation and transformation of two rare ginsenosides, Rg3 and Rh2. CN104894204 A discloses a method for preparing rare ginseng saponins by microbial enzymatic conversion of ginseng stem and leaf saponins. This method involves reacting an enzyme solution obtained from Aspergillus fermentation with ginseng stem and leaf protopylene saponins, American ginseng stem and leaf protopylene saponins, or Panax notoginseng stem and leaf saponins to prepare rare ginseng saponins C-Mx, C-Mc, CY, and CK. This invention uses Aspergillus, and the target compounds of the converted rare saponins are different. CN 105603036 A discloses a method for producing ginseng saponin Rh2, using Lactobacillus bulgaricus as the strain to ferment ginseng powder to produce Rh2. CN105648021 A discloses a method for preparing rare ginseng saponins CK, F1, and four isomers of ginsenosides. This patent mainly utilizes enzymes produced by the fermentation of Aspergillus niger and Aspergillus oryzae for treatment. CN105861381 A discloses a method for fermenting ginseng liquid using Lactobacillus plantarum. Compared with ordinary Lactobacillus plantarum, the antioxidant capacity and Rg3 and F2 content of the fermented ginseng liquid using a certain type of Lactobacillus plantarum are greatly improved.

[0006] In summary, there are currently many patents for ginseng fermentation, which mainly utilize lactic acid bacteria, yeast, Aspergillus niger, and Aspergillus oryzae for fermentation. Among them, yeast and lactic acid bacteria are edible fungi and have higher safety. Summary of the Invention

[0007] The existing technical problem is that the existing technology uses ginseng or single ginsenosides as raw materials, and uses yeast, lactic acid bacteria, Aspergillus oryzae, Aspergillus niger and other strains for fermentation or extracting enzymes for conversion. The types of rare saponins that can be converted are limited to a few common rare saponins.

[0008] In order to solve the above-mentioned technical problems, the inventors discovered that a multi-stage mixed fermentation of yeast and lactic acid bacteria can achieve the efficient conversion of various inherent ginsenosides into rare saponins.

[0009] Specifically, the present invention proposes the following technical solution:

[0010] On one hand, the present invention provides a ginsenoside composition containing 0.02-0.1 mg / mL of rare ginsenosides, 0.009-0.029 mg / mL of R-Rg3, 0.087-0.245 mg / mL of Rg5, and 0.013-0.044 mg / mL of CK.

[0011] Preferably, the above-mentioned ginsenoside composition contains rare ginsenosides S-RG3 0.04-0.060 mg / mL, R-Rg3 0.013-0.029 mg / mL, Rg5 0.129-0.241 mg / mL and CK 0.018-0.044 mg / mL.

[0012] Preferably, the above-mentioned ginsenoside composition contains rare ginsenosides Rg2 (0.14–0.45 mg / mL), F2 (0.028–0.032 mg / mL), F4 (0.066–0.085 mg / mL), Rh2 (0.007–0.015 mg / mL), Rh4 (0.042–0.055 mg / mL), Rk2 (0.002–0.004 mg / mL), Rh3 (0.001–0.002 mg / mL), and PPT (0.019–0.026 mg / mL).

[0013] Preferably, in the above-mentioned ginsenoside composition, Rg2 is S-Rg2 and / or R-Rg2; and Rh2 is S-Rh2 and / or R-Rh2.

[0014] On the other hand, the present invention also provides a ginseng fermentation product containing the ginsenoside composition described in any of the preceding paragraphs.

[0015] Preferably, the ginseng fermentation product mentioned above is derived from edible fungi fermentation, and more preferably, the edible fungi are selected from yeast and / or lactic acid bacteria.

[0016] On the other hand, the present invention also provides a method for preparing ginseng ferment, comprising the following steps:

[0017] Step (1): Mix the raw materials to be fermented;

[0018] Step (2): Inoculate the mixture obtained in step (1) with yeast or lactic acid bacteria for fermentation; preferably, the weight ratio of the yeast to the mixture is (0.5-10):100, more preferably (5-10):100; the weight ratio of the lactobacillus to the mixture is (0.5-10):100, more preferably (5-10):100;

[0019] Step (3): Sterilization;

[0020] Step (4): Inoculate with lactic acid bacteria or yeast for fermentation, provided that the strain used in step (4) is different from the strain used in step (2); preferably, the weight ratio of the yeast to the mixture is (0.5-10):100, more preferably (0.5-5):100; the weight ratio of the lactobacillus to the mixture is (0.5-10):100, more preferably (0.5-5):100;

[0021] Step (5): Filter to obtain fermentation filtrate;

[0022] Step (6): Sterilization;

[0023] Preferably, in step (2), yeast is introduced for fermentation.

[0024] Preferably, in the above preparation method, the raw materials to be fermented include ginseng, a carbon source, and a nitrogen source.

[0025] Preferably, in the above preparation method, the ginseng is selected from ginseng root, ginseng flower, ginseng berry, ginseng stem and leaf and / or ginseng extract; the carbon source is selected from glucose, fructose, sucrose and / or maltose; and the nitrogen source is selected from ammonium sulfate, ammonium chloride, casein and / or milk powder.

[0026] Preferably, in the above preparation method, the ginseng extract is obtained by water extraction of ginseng; preferably, the water extraction temperature is 80-100℃, more preferably 80-85℃.

[0027] Preferably, in the above preparation method, the yeast is selected from Saccharomyces cerevisiae, Pichia pastoris and / or Candida albicans; the lactic acid bacteria are selected from Lactobacillus pentosus, Bifidobacterium longum, Bifidobacterium breve, Lactobacillus acidophilus, Lactobacillus plantarum and / or Lactobacillus rhamnosus.

[0028] Preferably, in the above preparation method, yeast fermentation is carried out in a shaker, and lactic acid bacteria fermentation is carried out in an anaerobic or aerobic culture.

[0029] Preferably, in the above preparation method, the yeast fermentation temperature is 25-35℃, more preferably 28-30℃, and the lactic acid bacteria fermentation temperature is 30-37℃, more preferably 35-37℃.

[0030] Preferably, in the above preparation method, the yeast fermentation time is 48–240 hours, more preferably 96–192 hours; the lactic acid bacteria fermentation time is 48–240 hours, more preferably 96–240 hours.

[0031] Preferably, in the above preparation method, step (3) includes adjusting the pH to 5.5-7.5. More preferably, the pH is adjusted to 6-7.5 before lactic acid bacteria fermentation and the pH is adjusted to 6.5-7 before yeast fermentation.

[0032] On the other hand, the present invention also provides ginseng fermentation products prepared by the above-described preparation method.

[0033] On the other hand, the present invention also provides a food, medicine, health food or skin care product containing the ginseng ferment of the present invention.

[0034] On the other hand, the present invention also provides the application of the ginseng ferment of the present invention in the preparation of foods, medicines, health foods or skin care products that enhance immunity.

[0035] The beneficial effects of this invention include:

[0036] This invention uses ginseng and its growth byproducts as raw materials, making it natural and safe.

[0037] This method achieves the conversion of ginseng's inherent saponins into rare saponins through stepwise mixed fermentation of two edible fungi, yeast and lactic acid bacteria. Seventeen rare saponins were efficiently converted, and the total amount of rare saponins increased by 200%-300% compared with that before fermentation.

[0038] The method of this invention is simple and has a short cycle time. This method can achieve efficient conversion of ginseng and its by-products, improve the bioavailability of ginsenosides in ginseng and its growth by-products, and contribute to the comprehensive utilization and development of ginseng. This product can be applied to many industries such as food, pharmaceuticals, health foods, and skincare products, thereby enhancing the value of ginsenosides.

[0039] Strain Preservation Information

[0040] The strain of Saccharomyces cerevisiae A3.12 used in this invention was deposited at the China Center for Type Culture Collection (CCTCC) on December 11, 2017, with accession number CCTCC NO:M2017781. The deposit address is: Wuhan University, Wuhan, China, postal code: 430072; telephone: (027)-68754052.

[0041] The strain *Wickerhamomyces anomalus* C1.7 used in this invention was deposited on December 11, 2017, at the China Center for Type Culture Collection (CCTCC), accession number CCTCC NO: M2017782, address: Wuhan University, Wuhan, China, postcode: 430072; telephone: (027)-68754052.

[0042] The strain Pichia pastoris C1.8 used in this invention was deposited at the China Center for Type Culture Collection (CCTCC) on December 11, 2017, with accession number CCTCC NO:M2017780. The deposit address is: Wuhan University, Wuhan, China, postal code: 430072; telephone: (027)-68754052.

[0043] The strain used in this invention, *Bifidobacterium breve 001*, was deposited on October 25, 2017, at the China Center for Type Culture Collection (CCTCC), with accession number CCTCC NO: M2017626. The deposit address is: Wuhan University, Wuhan, China, postal code: 430072; telephone: (027)-68754052.

[0044] The strain *Pediococcus pentosaceus* 001 used in this invention was deposited on October 25, 2017, at the China Center for Type Culture Collection (CCTCC), accession number CCTCC NO: M2017625, address: Wuhan University, Wuhan, China, postcode: 430072; telephone: (027)-68754052.

[0045] The strain Lactobacillus plantarum 001 used in this invention was deposited at the China Center for Type Culture Collection (CCTCC) on October 25, 2017, with accession number CCTCC NO:M2017629. The deposit address is: Wuhan University, Wuhan, China, postal code: 430072; telephone: (027)-68754052.

[0046] The strain Lactobacillus rhamnosus 001 used in this invention was deposited at the China Center for Type Culture Collection (CCTCC) on October 25, 2017, with accession number CCTCC NO:M2017630. The deposit address is: Wuhan University, Wuhan, China, postal code: 430072; telephone: (027)-68754052.

[0047] The strain Lactobacillus acidophilus 001 used in this invention was deposited at the China Center for Type Culture Collection (CCTCC) on October 25, 2017, with accession number CCTCC NO: M2017628. The deposit address is: Wuhan University, Wuhan, China, postal code: 430072; telephone: (027)-68754052.

[0048] The strain Lactobacillus casei 001 used in this invention was deposited at the China Center for Type Culture Collection (CCTCC) on October 25, 2017, with accession number CCTCC NO: M2017631. The deposit address is: Wuhan University, Wuhan, China, postal code: 430072; telephone: (027)-68754052.

[0049] The strain Bifidobacterium longum 001 used in this invention was deposited at the China Center for Type Culture Collection (CCTCC) on October 25, 2017, with accession number CCTCC NO:M2017627. The deposit address is: Wuhan University, Wuhan, China, postal code: 430072; telephone: (027)-68754052.

[0050] The present invention and its beneficial technical effects will be described in detail below with reference to various specific embodiments. Detailed Implementation

[0051] For the terms "ginsenosides" and "rare saponins" used in this invention: Ginsenosides Ra, Rb, Rc, Rd, Re, Rf, and Rg1 are inherent saponins in ginseng, which are present in high amounts, accounting for more than 95% of the total saponins. After hydrolysis, inherent ginsenosides lose their coordinating sugar chains to yield ginsenosides Rh1, Rh2, F2, F1, CK, PPD, and PPT, which are present in very small amounts or almost non-existent in ginseng and are referred to as rare ginsenosides.

[0052] The purpose of this invention is to provide a stepwise compound fermentation process for converting ginseng's inherent saponins into rare saponins, and a ginseng fermentation liquid product with high rare saponin content, comprising: (1) preparation of ginseng powder or ginseng extract; (2) activation, purification, and large-scale cultivation of microbial strains; (3) mixing of raw materials; (4) inoculation; (5) yeast fermentation (lactic acid bacteria fermentation); (6) replenishment of culture medium; (7) lactic acid bacteria fermentation (yeast fermentation); (8) clarification treatment; (9) sterilization; and (10) detection. The content of rare ginseng saponins in the fermented product increases by 200-300% compared with that before fermentation.

[0053] During production, stepwise fermentation using different combinations of two edible microorganisms, yeast and lactic acid bacteria, is employed. After optimizing the fermentation process, 17 rare ginseng saponins, including S-Rg2, R-Rg2, F1, F4, Rk3, F2, Rh4, S-Rg3, R-Rg3, PPT, Rg5, CK, S-Rh2, R-Rh2, Rk2, Rh3, and PPD, are converted into their highest content. This process achieves a high conversion rate of these 17 rare ginseng saponins.

[0054] The raw materials of this invention are ginseng and ginseng extracts (including but not limited to ginseng root, ginseng flower, ginseng fruit, ginseng stem and leaf), nitrogen sources (including but not limited to ammonium sulfate, ammonium chloride, ammonium sulfate, casein, milk powder), carbon sources (including but not limited to glucose, fructose, sucrose, maltose, etc.), and yeasts (including but not limited to wine yeast, Pichia pastoris, Candida albicans), lactic acid bacteria (including but not limited to Lactobacillus pentosus, Bifidobacterium longum, Bifidobacterium breve, Lactobacillus acidophilus, Lactobacillus plantarum, Lactobacillus rhamnosus, Lactobacillus casei). The raw materials can be purchased from the market.

[0055] The preferred distributed fermentation method of the present invention includes the following steps:

[0056] 1) Yeast strain activation: Pick a loopful of Candida albicans (or other yeast strains mentioned above) colonies and place them in YPD liquid medium. Then place the medium in a shaker to activate the strain.

[0057] 2) Strain purification: The activated bacterial solution from the previous step is serially diluted and plated to obtain individual colonies.

[0058] 3) Strain expansion culture: Pick a single colony from the previous plate and inoculate it into YPD liquid medium. Incubate at 28°C on a shaker. When the OD value = 1.0, the fermentation broth is obtained (the strain is in the logarithmic phase, with a concentration of approximately 1 × 10⁻⁶). 8 (CFU / ml).

[0059] 4) Lactic acid bacteria culture: Pick a loopful of *Lactobacillus plantarum* (or other lactic acid bacteria strains mentioned above) and place it in MRS liquid medium for incubation and activation. Following the yeast expansion culture method described above, perform purification and expansion culture. Inoculate single colonies into MRS liquid medium and incubate at 37°C for anaerobic or aerobic culture. When the OD value reaches 1.0, the lactic acid bacteria fermentation broth is obtained (the strain is in the logarithmic phase, with a concentration of approximately 1 × 10⁻⁶). 8 (CFU / ml).

[0060] 5) Preparation of ginseng extract: Take dried ginseng (including but not limited to ginseng root, ginseng flower, ginseng fruit, ginseng stem and leaves), crush it through a 60-100 mesh sieve, add 10 times the volume of deionized water, heat in a water bath at 80-100℃ for 2-4 hours, centrifuge and filter to obtain filtrate, repeat the above extraction steps 1-2 times, combine the filtrates, and freeze-dry the filtrate.

[0061] 6) Raw material mixing: By weight, add 0.5-5 parts of the above ginseng extract or 1-10 parts of ginseng powder (60-100 mesh sieve) to 100 parts of deionized water, then add 0.5-10 parts of glucose and / or maltose, 0.5-10 parts of casein and / or 0.5-10 parts of ammonium sulfate, and sterilize at 60℃-121℃ for 5s-240min.

[0062] 7) Yeast inoculation and fermentation: Add 0.5-10 parts of the expanded culture of the bacterial solution to 100 parts of the cooled raw material mixture from the previous step, and incubate at 25℃-35℃ on a shaker for 48-240 hours (shaker speed 160-200 rpm / min).

[0063] 8) Sterilization adjustment: After the yeast fermentation is completed, add 0.5-10 parts of glucose, sucrose and / or maltose, 0.5-10 parts of casein, ammonium sulfate and / or ammonium sulfate, etc., then adjust the pH to between 5.5 and 7.5, and sterilize at 60℃-121℃ for 5s-240min.

[0064] 9) Lactic acid bacteria inoculation and fermentation: Add 0.5-10 parts of the expanded culture of lactic acid bacteria to 100 parts of the cooled yeast fermentation mixture from the previous step, and culture at 30-37℃ for anaerobic or aerobic 48-240h.

[0065] Another preferred stepwise fermentation method for steps 6) to 9) is: first, lactic acid bacteria fermentation is carried out according to the lactic acid bacteria fermentation process, and after fermentation, sterilization and adjustment are performed, and then yeast fermentation is carried out according to the yeast fermentation process.

[0066] 10) Clarification treatment: The fermentation products after fermentation are filtered through diatomaceous earth filtration or membrane filtration to obtain the supernatant, which is the ginseng fermentation liquid.

[0067] 11) Sterilization: The above ginseng fermentation liquid is sterilized at 60℃-115℃ for 5s-240min.

[0068] 13) Detection: The 17 rare ginsenosides, including S-Rg2, R-Rg2, F1, F4, Rk3, F2, Rh4, S-Rg3, R-Rg3, PPT, Rg5, CK, S-Rh2, R-Rh2, Rk2, Rh3, and PPD, were detected by HPLC analysis.

[0069] The specific detection and analysis steps using HPLC are as follows:

[0070] An Agilent 1200 high-performance liquid chromatograph was used with an Agilent Eclipse C18plus column (150 mm × 4.6 mm, 5 μm). The flow rate was acetonitrile (ACN)-0.1% phosphoric acid aqueous solution for gradient elution, as shown in Table 1. The flow rate was 1 mL / min, the column temperature was 30 °C, and the detection wavelength was 203 nm.

[0071] Table 1 Gradient Elution Table

[0072] Time ACN 0.1% Phosphoric acid water 0 19.5 80.5 25 19.5 80.5 40 32 68 60 33.5 66.5 65 38 62 70 62 38 75 62 38 90 65 35 95 95 5 100 95 5 100.1 19.5 80.5 105 19.5 80.5

[0073] Using ginsenoside standards as a reference, the ginsenosides were analyzed before and after fermentation using retention time for qualitative analysis and external standard method for quantitative analysis.

[0074] The fermentation method of the present invention and the high-content rare saponin ginseng fermentation liquid product obtained by fermentation are illustrated below through specific embodiments.

[0075] The sources of the reagents and instruments used in the following examples are as follows. Any reagents, instruments, or procedures not described herein are those that can be routinely determined by a person skilled in the art:

[0076] Table 2. Reagents and instruments used in the examples.

[0077]

[0078]

[0079] Experimental group 1: Preparation of ginseng fermentation liquid

[0080] Example 1

[0081] 1) Strain activation: Pick a loopful of Pichia pastoris colony and put it into YPD liquid medium, then place it in a shaker to activate the strain.

[0082] 2) Strain purification: The activated bacterial solution from the previous step is serially diluted and plated to obtain individual colonies.

[0083] 3) Expanding the culture of the strain: Select a single colony from the plate in the previous step and inoculate it into YPD liquid medium. Incubate in a shaker at 30°C. When the OD value is 1.0, the fermentation liquid of Pichia pastoris is obtained.

[0084] 4) Lactic acid bacteria activation culture: Pick a loopful of *Lactobacillus plantarum* colonies and place them in MRS liquid medium, then place the medium in an aerobic tank for activation culture. Following the yeast expansion culture procedure described above, perform purification and expansion culture, picking single colonies and inoculating them into MRS liquid medium. Incubate at 37°C with aerobic culture. When the OD value reaches 1.0, the *Lactobacillus plantarum* fermentation broth is obtained.

[0085] 5) Mixing raw materials: Take 10 parts ginseng root powder, 5 parts sucrose, 5 parts glucose, and 10 parts ammonium sulfate and add them to 100 parts deionized water. Sterilize at 60℃ for 2 hours.

[0086] 6) Yeast fermentation: Take 10 portions of the expanded cultured Pichia pastoris liquid and add it to the cooled raw material mixture from the previous step. Incubate at 28°C on a shaker for 96 hours (shaker speed 180 rpm / min).

[0087] 7) Sterilization adjustment: After the yeast fermentation is completed, add 5 parts of sucrose to adjust the pH to 7.5, and sterilize at 120℃ for 15 seconds.

[0088] 8) Lactic acid bacteria inoculation and fermentation: Add 5 portions of expanded cultured Lactobacillus plantarum liquid to the raw material mixture after cooling in the previous step, and culture at 37°C with oxygen for 144 hours.

[0089] 9) Clarification treatment: After fermentation, the supernatant obtained by fine filtration is the ginseng fermentation filtrate.

[0090] 10) Sterilization: Sterilize the above ginseng fermentation liquid at 100℃ for 30 minutes.

[0091] 11) Detection: The 17 rare ginsenosides, including S-Rg2, R-Rg2, F1, F4, Rk3, F2, Rh4, S-Rg3, R-Rg3, PPT, Rg5, CK, S-Rh2, R-Rh2, Rk2, Rh3, and PPD, were detected by HPLC. The results are shown in Table 3. The total amount of rare ginsenosides obtained in this example increased by 338% compared with the total amount of rare ginsenosides before fermentation.

[0092] Table 3. Ginsenoside content and conversion rate before and after fermentation in Example 1.

[0093]

[0094]

[0095] Since Rh3 was not detected before fermentation but reached 0.002 after fermentation, its conversion rate was positive infinity. Therefore, it was not included in the total rare saponin conversion rate for ease of comparison.

[0096] Example 2

[0097] 1) Activation of lactic acid bacteria strains: Pick a loopful of Lactobacillus pentosus colonies and put them into MRS liquid medium, then place them in an anaerobic tank for activation culture.

[0098] 2) Strain purification: The activated bacterial solution from the previous step is serially diluted and plated to obtain individual colonies.

[0099] 3) Expanding the bacterial culture: Select a single colony from the plate in the previous step and inoculate it into MRS liquid medium. Incubate anaerobically at 37°C. When the OD value is 1.0, the fermentation bacterial broth is obtained.

[0100] 4) Yeast activation culture: Pick a loopful of Candida albicans colony and place it in YPD liquid medium. Place the medium in a shaker to activate the culture. Following the above method for expanding lactic acid bacteria culture, perform purification and expansion culture. Pick a single colony and inoculate it into YPD liquid medium. Incubate at 30°C in a shaker. When the OD value = 1.0, the fermentation broth is obtained.

[0101] 5) Mixing raw materials: Take 5 parts ginseng fruit powder, 5 parts glucose, and 5 parts ammonium sulfate and add them to 100 parts deionized water. Sterilize at 121℃ for 15 minutes.

[0102] 6) Lactic acid bacteria inoculation and fermentation: Add 10 portions of the expanded culture of Lactobacillus pentosus to the raw material mixture after cooling in the previous step, and anaerobic culture at 37°C for 96 hours.

[0103] 7) Sterilization adjustment: After the lactic acid bacteria fermentation is completed, add 5 parts glucose, 10 parts ammonium sulfate and 2 parts milk powder to adjust the pH to 7.0, and sterilize at 60℃ for 60 minutes.

[0104] 8) Yeast fermentation: Take 10 portions of the expanded culture of Candida albicans and add them to the raw material mixture after cooling in the previous step. Incubate at 30°C on a shaker for 144 hours (shaker speed 180 rpm / min).

[0105] 9) Clarification treatment: After fermentation, the supernatant obtained by fine filtration is the ginseng fermentation filtrate.

[0106] 10) Sterilization: Sterilize the above ginseng fermentation liquid at 120℃ for 15 seconds.

[0107] 11) Detection: The 17 rare ginseng saponins, including S-Rg2, R-Rg2, F1, F4, Rk3, F2, Rh4, S-Rg3, R-Rg3, PPT, Rg5, CK, S-Rh2, R-Rh2, Rk2, Rh3, and PPD, were detected by HPLC. The total rare saponin content before fermentation in this example was 0.128 mg / ml, and the total rare saponin content after fermentation was 0.410 mg / ml. The total rare saponin content of the obtained ginseng was increased by 220% compared with that before fermentation. After fermentation, the content of S-Rg3 was 0.04 mg / ml, the content of R-Rg3 was 0.013 mg / ml, the content of Rg5 was 0.129 mg / ml, and the content of CK was 0.018 mg / ml.

[0108] Example 3

[0109] 1) Strain activation: Pick a loopful of Saccharomyces cerevisiae colony and put it into YPD liquid medium, then place it in a shaker to activate the strain.

[0110] 2) Strain purification: The activated bacterial solution from the previous step is serially diluted and plated to obtain individual colonies.

[0111] 3) Expanding the culture of the strain: Pick a single colony from the plate in the previous step and inoculate it into YPD liquid medium. Incubate in a shaker at 28°C. When the OD value is 1.0, the fermentation broth is obtained.

[0112] 4) Lactic acid bacteria activation culture: Pick a loopful of Bifidobacterium breve colonies and place them in MRS liquid medium, then place the medium in an anaerobic tank for activation culture. Following the yeast expansion culture procedure described above, perform purification and expansion culture, picking single colonies and inoculating them into MRS liquid medium. Incubate anaerobically at 37°C. When the OD value reaches 1.0, the fermentation broth is obtained.

[0113] 5) Ginseng extract: Mix 10 parts of ginseng root powder with 100 parts of deionized water, heat in a water bath at 80°C for 4 hours, centrifuge and filter to obtain filtrate, repeat the above extraction steps twice, combine the filtrates, freeze dry the filtrate to obtain lyophilized ginseng extract powder.

[0114] 6) Mixing raw materials: Take 1 part of the above freeze-dried powder, 5 parts of sucrose, 10 parts of milk powder and 5 parts of ammonium sulfate and add them to 100 parts of deionized water. Sterilize at 115℃ for 15 minutes.

[0115] 7) Yeast fermentation: Take 5 portions of the expanded yeast culture and add them to the cooled raw material mixture in step 6), and incubate at 25°C on a shaker for 96 hours (shaker speed 160 rpm / min).

[0116] 8) Sterilization adjustment: After the yeast fermentation is completed, add 5 parts of sucrose to adjust the pH to 6.5, and sterilize at 100℃ for 30 minutes.

[0117] 9) Lactic acid bacteria inoculation and fermentation: Add 5 portions of the expanded cultured lactic acid bacteria liquid to the cooled raw material mixture from the previous step, and culture at 37°C for 144 hours in anaerobic or aerobic conditions.

[0118] 10) Clarification treatment: After fermentation, the supernatant obtained by fine filtration is the ginseng fermentation filtrate.

[0119] 11) Sterilization: Sterilize the above ginseng fermentation liquid at 60℃ for 1 hour.

[0120] 12) Detection: The 17 rare ginseng saponins, including S-Rg2, R-Rg2, F1, F4, Rk3, F2, Rh4, S-Rg3, R-Rg3, PPT, Rg5, CK, S-Rh2, R-Rh2, Rk2, Rh3, and PPD, were detected by HPLC. The total rare saponin content before fermentation in this example was 0.18 mg / ml, and the total rare saponin content after fermentation was 0.62 mg / ml. The total rare saponin content of the obtained ginseng saponins increased by 244% compared with that before fermentation. After fermentation, the content of S-Rg3 was 0.057 mg / ml, the content of R-Rg3 was 0.025 mg / ml, the content of Rg5 was 0.206 mg / ml, and the content of CK was 0.034 mg / ml.

[0121] Example 4

[0122] 1) Activation of lactic acid bacteria strains: Pick a loopful of Lactobacillus pentosus colonies and put them into MRS liquid medium, then place them in an anaerobic tank for activation culture.

[0123] 2) Strain purification: The activated bacterial solution from the previous step is serially diluted and plated to obtain individual colonies.

[0124] 3) Expanding the bacterial culture: Select a single colony from the plate in the previous step and inoculate it into MRS liquid medium. Incubate anaerobically at 37°C. When the OD value is 1.0, the fermentation bacterial broth is obtained.

[0125] 4) Yeast activation culture: Pick a loopful of Candida albicans colony and place it in YPD liquid medium. Place the medium in a shaker to activate the culture. Following the above method for expanding lactic acid bacteria culture, perform purification and expansion culture. Pick a single colony and inoculate it into YPD liquid medium. Incubate at 30°C in a shaker. When the OD value = 1.0, the fermentation broth is obtained.

[0126] 5) Mixing raw materials: Take 5 parts ginseng fruit powder, 5 parts glucose, and 5 parts ammonium sulfate and add them to 100 parts deionized water. Sterilize at 121℃ for 15 minutes.

[0127] 6) Lactic acid bacteria inoculation and fermentation: Add 10 portions of the expanded culture of Lactobacillus pentosus to the raw material mixture after cooling in the previous step, and culture anaerobically at 30°C for 240 hours.

[0128] 7) Sterilization adjustment: After the lactic acid bacteria fermentation is completed, add 5 parts glucose, 10 parts ammonium sulfate and 2 parts milk powder to adjust the pH to 7.0, and sterilize at 60℃ for 60 minutes.

[0129] 8) Yeast fermentation: Take 10 portions of the expanded culture of Candida albicans and add them to the raw material mixture after cooling in the previous step. Incubate at 35°C on a shaker for 48 hours (shaker speed 180 rpm / min).

[0130] 9) Clarification treatment: After fermentation, the supernatant obtained by fine filtration is the ginseng fermentation filtrate.

[0131] 10) Sterilization: Sterilize the above ginseng fermentation liquid at 120℃ for 15 seconds.

[0132] 11) Detection: The 17 rare ginseng saponins, including S-Rg2, R-Rg2, F1, F4, Rk3, F2, Rh4, S-Rg3, R-Rg3, PPT, Rg5, CK, S-Rh2, R-Rh2, Rk2, Rh3, and PPD, were detected by HPLC. The total rare saponin content before fermentation in this example was 0.132 mg / ml, and the total rare saponin content after fermentation was 0.489 mg / ml. The total rare saponin content of the obtained ginseng saponins increased by 270% compared with that before fermentation. After fermentation, the content of S-Rg3 was 0.10 mg / ml, the content of R-Rg3 was 0.012 mg / ml, the content of Rg5 was 0.140 mg / ml, and the content of CK was 0.025 mg / ml.

[0133] Example 5

[0134] 1) Activation of lactic acid bacteria strains: Pick a loopful of Lactobacillus rhamnosus colonies and put them into MRS liquid medium, then place them in an anaerobic tank for activation culture.

[0135] 2) Strain purification: The activated bacterial solution from the previous step is serially diluted and plated to obtain individual colonies.

[0136] 3) Expanding the bacterial culture: Select a single colony from the plate in the previous step and inoculate it into MRS liquid medium. Incubate anaerobically at 37°C. When the OD value is 1.0, the fermentation bacterial broth is obtained.

[0137] 4) Yeast activation culture: Pick a loopful of Candida albicans colony and place it in YPD liquid medium. Place the medium in a shaker to activate the culture. Following the above method for expanding lactic acid bacteria culture, perform purification and expansion culture. Pick a single colony and inoculate it into YPD liquid medium. Incubate at 30°C in a shaker. When the OD value = 1.0, the fermentation broth is obtained.

[0138] 5) Mixing raw materials: Take 5 parts ginseng fruit powder, 5 parts glucose, and 5 parts ammonium sulfate and add them to 100 parts deionized water. Sterilize at 121℃ for 15 minutes.

[0139] 6) Lactic acid bacteria inoculation and fermentation: Add 10 portions of the expanded culture of Lactobacillus rhamnosus to the raw material mixture after cooling in the previous step, and anaerobic culture at 34℃ for 48h.

[0140] 7) Sterilization adjustment: After the lactic acid bacteria fermentation is completed, add 5 parts glucose, 10 parts ammonium sulfate and 2 parts milk powder to adjust the pH to 7.0, and sterilize at 60℃ for 60 minutes.

[0141] 8) Yeast fermentation: Take 10 portions of the expanded culture of Candida albicans and add them to the raw material mixture after cooling in the previous step. Incubate at 30°C on a shaker for 96 hours (shaker speed 180 rpm / min).

[0142] 9) Clarification treatment: After fermentation, the supernatant obtained by fine filtration is the ginseng fermentation filtrate.

[0143] 10) Sterilization: Sterilize the above ginseng fermentation liquid at 120℃ for 15 seconds.

[0144] 11) Detection: The 17 rare ginseng saponins, including S-Rg2, R-Rg2, F1, F4, Rk3, F2, Rh4, S-Rg3, R-Rg3, PPT, Rg5, CK, S-Rh2, R-Rh2, Rk2, Rh3, and PPD, were detected by HPLC. The total rare saponin content before fermentation in this example was 0.098 mg / ml, and the total rare saponin content after fermentation was 0.420 mg / ml. The total rare saponin content of the obtained ginseng saponins increased by 329% compared with that before fermentation. After fermentation, the content of S-Rg3 was 0.06 mg / ml, the content of R-Rg3 was 0.023 mg / ml, the content of Rg5 was 0.152 mg / ml, and the content of CK was 0.028 mg / ml.

[0145] Example 6

[0146] 1) Activation of lactic acid bacteria strains: Pick a loopful of Lactobacillus acidophilus colonies and put them into MRS liquid medium, then place them in an anaerobic tank for activation culture.

[0147] 2) Strain purification: The activated bacterial solution from the previous step is serially diluted and plated to obtain individual colonies.

[0148] 3) Expanding the bacterial culture: Select a single colony from the plate in the previous step and inoculate it into MRS liquid medium. Incubate anaerobically at 37°C. When the OD value is 1.0, the fermentation bacterial broth is obtained.

[0149] 4) Yeast activation culture: Pick a loopful of Candida albicans colony and place it in YPD liquid medium. Place the medium in a shaker to activate the culture. Following the above method for expanding lactic acid bacteria culture, perform purification and expansion culture. Pick a single colony and inoculate it into YPD liquid medium. Incubate at 30°C in a shaker. When the OD value = 1.0, the fermentation broth is obtained.

[0150] 5) Mixing raw materials: Take 5 parts ginseng fruit powder, 5 parts glucose, and 5 parts ammonium sulfate and add them to 100 parts deionized water. Sterilize at 121℃ for 15 minutes.

[0151] 6) Lactic acid bacteria inoculation and fermentation: Add 5 portions of expanded cultured Lactobacillus acidophilus liquid to the raw material mixture after cooling in the previous step, and anaerobic culture at 37°C for 96 hours.

[0152] 7) Sterilization adjustment: After the lactic acid bacteria fermentation is completed, add 5 parts glucose, 10 parts ammonium sulfate and 2 parts milk powder to adjust the pH to 7.0, and sterilize at 60℃ for 60 minutes.

[0153] 8) Yeast fermentation: Take 0.5 parts of the expanded culture of Candida albicans and add it to the raw material mixture after cooling in the previous step. Incubate at 30°C on a shaker for 144 hours (shake speed 180 rpm / min).

[0154] 9) Clarification treatment: After fermentation, the supernatant obtained by fine filtration is the ginseng fermentation filtrate.

[0155] 10) Sterilization: Sterilize the above ginseng fermentation liquid at 120℃ for 15 seconds.

[0156] 11) Detection: The 17 rare ginseng saponins, including S-Rg2, R-Rg2, F1, F4, Rk3, F2, Rh4, S-Rg3, R-Rg3, PPT, Rg5, CK, S-Rh2, R-Rh2, Rk2, Rh3, and PPD, were detected by HPLC. The total rare saponin content before fermentation in this example was 0.110 mg / ml, and the total rare saponin content after fermentation was 0.409 mg / ml. The total rare saponin content of the obtained ginseng saponins increased by 272% compared with that before fermentation. After fermentation, the content of S-Rg3 was 0.05 mg / ml, the content of R-Rg3 was 0.017 mg / ml, the content of Rg5 was 0.112 mg / ml, and the content of CK was 0.016 mg / ml.

[0157] Example 7

[0158] 1) Strain activation: Pick a loopful of Saccharomyces cerevisiae colony and put it into YPD liquid medium, then place it in a shaker to activate the strain.

[0159] 2) Strain purification: The activated bacterial solution from the previous step is serially diluted and plated to obtain individual colonies.

[0160] 3) Expanding the culture of the strain: Pick a single colony from the plate in the previous step and inoculate it into YPD liquid medium. Incubate in a shaker at 28°C. When the OD value is 1.0, the fermentation broth is obtained.

[0161] 4) Lactic acid bacteria activation culture: Pick a loopful of Lactobacillus casei colony and place it in MRS liquid medium, then place it in an anaerobic tank for activation culture. Following the yeast expansion culture procedure described above, perform purification and expansion culture, picking a single colony and inoculating it into MRS liquid medium. Incubate anaerobicly at 37℃. When the OD value reaches 1.0, the fermentation broth is obtained.

[0162] 5) Ginseng extract: Mix 10 parts of ginseng root powder with 100 parts of deionized water, heat in a water bath at 80°C for 4 hours, centrifuge and filter to obtain filtrate, repeat the above extraction steps twice, combine the filtrates, freeze dry the filtrate to obtain lyophilized ginseng extract powder.

[0163] 6) Mixing raw materials: Take 1 part of the above freeze-dried powder, 5 parts of sucrose, 10 parts of milk powder and 5 parts of ammonium sulfate and add them to 100 parts of deionized water. Sterilize at 115℃ for 15 minutes.

[0164] 7) Yeast fermentation: Take 5 portions of the expanded yeast culture and add them to the cooled raw material mixture in step 6), and culture at 30°C on a shaker for 192 hours (shaker speed 160 rpm / min).

[0165] 8) Sterilization adjustment: After the yeast fermentation is completed, add 5 parts of sucrose to adjust the pH to 6.0, and sterilize at 100℃ for 30 minutes.

[0166] 9) Lactic acid bacteria inoculation and fermentation: Add 5 portions of the expanded cultured lactic acid bacteria liquid to the raw material mixture after cooling in the previous step, and culture at 30°C for 144 hours in anaerobic or aerobic conditions.

[0167] 10) Clarification treatment: After fermentation, the supernatant obtained by fine filtration is the ginseng fermentation filtrate.

[0168] 11) Sterilization: Sterilize the above ginseng fermentation liquid at 60℃ for 1 hour.

[0169] 12) Detection: The 17 rare ginseng saponins, including S-Rg2, R-Rg2, F1, F4, Rk3, F2, Rh4, S-Rg3, R-Rg3, PPT, Rg5, CK, S-Rh2, R-Rh2, Rk2, Rh3, and PPD, were detected by HPLC. The total rare saponin content before fermentation in this example was 0.15 mg / ml, and the total rare saponin content after fermentation was 0.68 mg / ml. The total rare saponin content of the obtained ginseng saponins increased by 353% compared with that before fermentation. The content of S-Rg3 after fermentation was 0.067 mg / ml, the content of R-Rg3 was 0.028 mg / ml, the content of Rg5 was 0.222 mg / ml, and the content of CK was 0.038 mg / ml.

[0170] Example 8

[0171] 1) Strain activation: Pick a loopful of Saccharomyces cerevisiae colony and put it into YPD liquid medium, then place it in a shaker to activate the strain.

[0172] 2) Strain purification: The activated bacterial solution from the previous step is serially diluted and plated to obtain individual colonies.

[0173] 3) Expanding the culture of the strain: Pick a single colony from the plate in the previous step and inoculate it into YPD liquid medium. Incubate in a shaker at 28°C. When the OD value is 1.0, the fermentation broth is obtained.

[0174] 4) Lactic acid bacteria activation culture: Pick a loopful of Bifidobacterium longum colony and place it in MRS liquid medium, then place it in an anaerobic tank for activation culture. Following the yeast expansion culture procedure described above, perform purification and expansion culture, picking a single colony and inoculating it into MRS liquid medium. Incubate anaerobicly at 37℃. When the OD value reaches 1.0, the fermentation broth is obtained.

[0175] 5) Ginseng extract: Mix 10 parts of ginseng root powder with 100 parts of deionized water, heat in a water bath at 80°C for 4 hours, centrifuge and filter to obtain filtrate, repeat the above extraction steps twice, combine the filtrates, freeze dry the filtrate to obtain lyophilized ginseng extract powder.

[0176] 6) Mixing raw materials: Take 1 part of the above freeze-dried powder, 5 parts of sucrose, 10 parts of milk powder and 5 parts of ammonium sulfate and add them to 100 parts of deionized water. Sterilize at 115℃ for 15 minutes.

[0177] 7) Yeast fermentation: Take 5 portions of the expanded yeast culture and add them to the cooled raw material mixture in step 6), and incubate at 35°C on a shaker for 48 hours (shaker speed 160 rpm / min).

[0178] 8) Sterilization adjustment: After the yeast fermentation is completed, add 5 parts of sucrose to adjust the pH to 6.0, and sterilize at 100℃ for 30 minutes.

[0179] 9) Lactic acid bacteria inoculation and fermentation: Add 5 portions of the expanded cultured lactic acid bacteria liquid to the cooled raw material mixture from the previous step, and culture at 32°C for 192 hours in anaerobic or aerobic conditions.

[0180] 10) Clarification treatment: After fermentation, the supernatant obtained by fine filtration is the ginseng fermentation filtrate.

[0181] 11) Sterilization: Sterilize the above ginseng fermentation liquid at 60℃ for 1 hour.

[0182] 12) Detection: The 17 rare ginseng saponins, including S-Rg2, R-Rg2, F1, F4, Rk3, F2, Rh4, S-Rg3, R-Rg3, PPT, Rg5, CK, S-Rh2, R-Rh2, Rk2, Rh3, and PPD, were detected by HPLC. The total rare saponin content before fermentation in this example was 0.17 mg / ml, and the total rare saponin content after fermentation was 0.58 mg / ml. The total rare saponin content of the obtained ginseng saponins increased by 296% compared with that before fermentation. After fermentation, the content of S-Rg3 was 0.052 mg / ml, the content of R-Rg3 was 0.032 mg / ml, the content of Rg5 was 0.211 mg / ml, and the content of CK was 0.039 mg / ml.

[0183] Example 9

[0184] 1) Strain activation: Pick a loopful of Saccharomyces cerevisiae colony and put it into YPD liquid medium, then place it in a shaker to activate the strain.

[0185] 2) Strain purification: The activated bacterial solution from the previous step is serially diluted and plated to obtain individual colonies.

[0186] 3) Expanding the culture of the strain: Pick a single colony from the plate in the previous step and inoculate it into YPD liquid medium. Incubate in a shaker at 28°C. When the OD value is 1.0, the fermentation broth is obtained.

[0187] 4) Lactic acid bacteria activation culture: Pick a loopful of Bifidobacterium breve colonies and place them in MRS liquid medium, then place the medium in an anaerobic tank for activation culture. Following the yeast expansion culture procedure described above, perform purification and expansion culture, picking single colonies and inoculating them into MRS liquid medium. Incubate anaerobically at 37°C. When the OD value reaches 1.0, the fermentation broth is obtained.

[0188] 5) Ginseng extract: Mix 10 parts of ginseng root powder with 100 parts of deionized water, heat in a water bath at 80°C for 4 hours, centrifuge and filter to obtain filtrate, repeat the above extraction steps twice, combine the filtrates, freeze dry the filtrate to obtain lyophilized ginseng extract powder.

[0189] 6) Mixing raw materials: Take 1 part of the above freeze-dried powder, 5 parts of sucrose, 10 parts of milk powder and 5 parts of ammonium sulfate and add them to 100 parts of deionized water. Sterilize at 115℃ for 15 minutes.

[0190] 7) Yeast fermentation: Take 10 portions of the expanded yeast culture and add them to the cooled raw material mixture in step 6), and incubate at 25°C on a shaker for 96 hours (shaker speed 160 rpm / min).

[0191] 8) Sterilization adjustment: After the yeast fermentation is completed, add 5 parts of sucrose to adjust the pH to 6.0, and sterilize at 100℃ for 30 minutes.

[0192] 9) Lactic acid bacteria inoculation and fermentation: Add 0.5 parts of the expanded culture of lactic acid bacteria to the raw material mixture after cooling in the previous step, and culture at 37°C for 144 hours in anaerobic or aerobic conditions.

[0193] 10) Clarification treatment: After fermentation, the supernatant obtained by fine filtration is the ginseng fermentation filtrate.

[0194] 11) Sterilization: Sterilize the above ginseng fermentation liquid at 60℃ for 1 hour.

[0195] 12) Detection: The 17 rare ginseng saponins, including S-Rg2, R-Rg2, F1, F4, Rk3, F2, Rh4, S-Rg3, R-Rg3, PPT, Rg5, CK, S-Rh2, R-Rh2, Rk2, Rh3, and PPD, were detected by HPLC. The total rare saponin content before fermentation in this example was 0.15 mg / ml, and the total rare saponin content after fermentation was 0.67 mg / ml. The total rare saponin content of the obtained ginseng saponins increased by 347% compared with that before fermentation. After fermentation, the content of S-Rg3 was 0.052 mg / ml, the content of R-Rg3 was 0.029 mg / ml, the content of Rg5 was 0.241 mg / ml, and the content of CK was 0.044 mg / ml.

[0196] Comparative Example 1

[0197] Fermentation is carried out using a mixed fermentation method:

[0198] 1) Strain activation: Pick a loopful of Candida albicans colony and put it into YPD liquid medium, then place it in a shaker to activate the strain.

[0199] 2) Strain purification: The activated bacterial solution from the previous step is serially diluted and plated to obtain individual colonies.

[0200] 3) Expanding the culture of the strain: Select a single colony from the plate in the previous step and inoculate it into YPD liquid medium. Incubate in a shaker at 30°C. When the OD value is 1.0, the fermentation liquid of Pichia pastoris is obtained.

[0201] 4) Lactic acid bacteria activation culture: Pick a loopful of *Lactobacillus plantarum* colonies and place them in MRS liquid medium, then place the medium in an aerobic tank for activation culture. Following the yeast expansion culture procedure described above, perform purification and expansion culture, picking single colonies and inoculating them into MRS liquid medium. Incubate at 37°C with aerobic culture. When the OD value reaches 1.0, the *Lactobacillus plantarum* fermentation broth is obtained.

[0202] 5) Mixing raw materials: Take 10 parts ginseng root powder, 5 parts sucrose, 5 parts glucose, and 10 parts ammonium sulfate and add them to 100 parts deionized water. Sterilize at 60℃ for 2 hours.

[0203] 6) Mixed fermentation: Take 10 parts of the expanded culture of Pichia pastoris and 10 parts of Lactobacillus plantarum fermentation bacteria and add them to the raw material mixture after cooling in the previous step. Incubate at 30°C on a shaker for 120 hours (shaker speed 100 rpm / min).

[0204] 9) Clarification treatment: After fermentation, the supernatant obtained by membrane filtration is the ginseng fermentation filtrate.

[0205] 10) Sterilization: Sterilize the above ginseng fermentation liquid at 100℃ for 30 minutes.

[0206] 11) Detection: The 17 rare ginseng saponins, including S-Rg2, R-Rg2, F1, F4, Rk3, F2, Rh4, S-Rg3, R-Rg3, PPT, Rg5, CK, S-Rh2, R-Rh2, Rk2, Rh3, and PPD, were detected by HPLC. The total rare saponin content before fermentation in this example was 0.15 mg / ml, and the total rare saponin content after fermentation was 0.166 mg / ml. The total rare saponin content of the obtained ginseng was increased by 104% compared with that before fermentation.

[0207] Comparative Example 2

[0208] Yeast fermentation

[0209] 1) Strain activation: Pick a loopful of Saccharomyces cerevisiae colony and put it into YPD liquid medium, then place it in a shaker to activate the strain.

[0210] 2) Strain purification: The activated bacterial solution from the previous step is serially diluted and plated to obtain individual colonies.

[0211] 3) Expanding the culture of the strain: Pick a single colony from the plate in the previous step and inoculate it into YPD liquid medium. Incubate in a shaker at 28°C. When the OD value is 1.0, the fermentation broth is obtained.

[0212] 5) Ginseng extract: Mix 10 parts of ginseng root powder with 100 parts of deionized water, heat in a water bath at 80°C for 4 hours, centrifuge and filter to obtain filtrate, repeat the above extraction steps twice, combine the filtrates, and freeze-dry the filtrate.

[0213] 6) Mixing raw materials: Take 1 part of the above freeze-dried powder, 5 parts of sucrose, 10 parts of milk powder and 5 parts of ammonium sulfate and add them to 100 parts of deionized water. Sterilize at 115℃ for 15 minutes.

[0214] 7) Yeast fermentation: Take 5 portions of the expanded yeast culture and add them to the cooled raw material mixture from the previous step. Incubate at 25°C on a shaker for 96 hours (shaker speed 160 rpm / min).

[0215] 8) Clarification treatment: After fermentation, the supernatant obtained by plate and frame filtration is the ginseng fermentation filtrate.

[0216] 9) Sterilization: Sterilize the above ginseng fermentation liquid at 60℃ for 1 hour.

[0217] 10) Detection: The 17 rare ginseng saponins, including S-Rg2, R-Rg2, F1, F4, Rk3, F2, Rh4, S-Rg3, R-Rg3, PPT, Rg5, CK, S-Rh2, R-Rh2, Rk2, Rh3, and PPD, were detected by HPLC. The total rare saponin content before fermentation in this example was 0.145 mg / ml, and the total rare saponin content after fermentation was 0.252 mg / ml. The total rare saponin content of the obtained ginseng was increased by 74% compared with that before fermentation.

[0218] Comparative Example 3

[0219] Fermentation using Bifidobacterium breve

[0220] 1) Lactic acid bacteria activation culture: Pick a loopful of Bifidobacterium breve colonies and place them in MRS liquid medium, then place the medium in an anaerobic tank for activation culture. Following the yeast expansion culture procedure described above, perform purification and expansion culture, picking single colonies and inoculating them into MRS liquid medium. Incubate anaerobically at 37°C. When the OD value reaches 1.0, the fermentation broth is obtained.

[0221] 2) Ginseng extract: Mix 10 parts of ginseng root powder with 100 parts of deionized water, heat in a water bath at 80°C for 4 hours, centrifuge and filter to obtain filtrate, repeat the above extraction steps twice, combine the filtrates, and freeze-dry the filtrate.

[0222] 3) Mixing raw materials: Take 1 part of the above freeze-dried powder, 5 parts of sucrose, 10 parts of milk powder and 5 parts of ammonium sulfate and add them to 100 parts of deionized water. Sterilize at 115℃ for 15 minutes.

[0223] 4) Lactic acid bacteria inoculation and fermentation: Add 5 portions of the expanded cultured lactic acid bacteria liquid to the cooled raw material mixture from the previous step, and culture at 37°C for 144 hours in anaerobic or aerobic conditions.

[0224] 5) Clarification treatment: After fermentation, the supernatant obtained by fine filtration is the ginseng fermentation filtrate.

[0225] 6) Sterilization: Sterilize the above ginseng fermentation liquid at 60℃ for 1 hour.

[0226] 7) Detection: HPLC analysis was used to detect 17 rare ginseng saponins, including S-Rg2, R-Rg2, F1, F4, Rk3, F2, Rh4, S-Rg3, R-Rg3, PPT, Rg5, CK, S-Rh2, R-Rh2, Rk2, Rh3, and PPD. The total rare saponin content before fermentation in this example was 0.16 mg / ml, and after fermentation, it was 0.365 mg / ml, representing a 128% increase in the total rare ginseng saponins obtained compared to before fermentation.

[0227] Experimental group 2: Comparison of natural killer (NK) cell viability in mice

[0228] Mouse treatment:

[0229] Eighteen mice were randomly divided into three groups of six each. The mice were administered ginseng extract (as described in Example 3) and ginseng fermentation broth (prepared in Example 3) by gavage at a dose of 10 g / kg body weight. The control group received the same dose of sterile water daily by gavage. After 20 days of continuous gavage administration, spleen cells were collected one hour after administration to prepare a spleen cell suspension (effect cells) with a concentration of 3.0 × 10⁻⁶ cells / kg. 6 per ml.

[0230] Experimental steps:

[0231] The cell concentration obtained from passage culture was 4×10⁻⁶. 5 100 μl each of target cells (YAC-1 cells) and effector cells (effector-target ratio 50:1) were added to a 96-well culture plate. 100 μl each of target cells and effector cells were added to the sample reaction wells. 100 μl each of target cells and culture medium were added to the target cell spontaneous release wells. 100 μl each of target cells, 1% NP40, and 2.5% Triton were added to the target cell maximum release wells. The plates were incubated at 37°C and 5% CO2 for 4 h. After centrifugation, 100 μl of supernatant was transferred from each well to another 96-well culture plate, along with 100 μl of LDH matrix solution. The reaction was allowed to proceed for 5 min. 30 μl of 1 mol / L HCl was added to each well, and the OD value was measured at 490 nm using a microplate reader. Three replicates were prepared for each well, as shown in Table 4.

[0232] Table 4

[0233] Sample reaction well Natural release well Maximum release well Target cell (μL) 100 100 100 Effector cell (μL) 100 Culture solution (μL) 100 2.5% Triton (μL) 100 1% NP40 (μL) 100 Supernatant (μL) 100 100 100 LDH substrate solution (μL) 100 100 100 1 mol / L HCL (μL) 30 30 30

[0234] NK cell activity is calculated using the following formula:

[0235] NK cell activity (%) = (OD of sample reaction wells - OD of spontaneous release wells) / (OD of maximum release wells - OD of spontaneous release wells) × 100%

[0236] The effects of ginseng on NK cell activity were analyzed and are shown in the table below:

[0237] Effects of ginseng on NK cell viability (n=6)

[0238] Group NK cell activity (%) Blank control group 28.9±9.11 Ginseng extract group 60.2±10.13 Ginseng fermentation broth group 78.9±10.99

[0239] Where: n=6 represents the experimental average of 6 mice in each group.

[0240] As can be seen from the table above, the cell activity of the ginseng fermentation sample group was significantly higher than that of the ginseng group and the blank control group. This is because the ginseng fermentation sample group had a higher content of rare saponins. The contents of rare saponins such as S-Rg2, R-Rg2, F4, F2, Rh4, S-Rg3, R-Rg3, PPT, Rg5, CK, S-Rh2, R-Rh2, Rk2, and Rh3 were all significantly increased compared with those before fermentation, which in turn significantly improved the cell activity of the ginseng fermentation sample group.

[0241] As can be seen from the above specific embodiments, the stepwise fermentation method of the present invention has a significant effect on the conversion of saponins compared with the mixed fermentation method or the single-strain fermentation method. The stepwise fermentation method of the present invention yields a rich variety of rare saponins, up to 17 kinds, and the total rare saponins are increased by 200-300% compared with those before fermentation. The fermented product has a significant effect on improving NK cell activity. Therefore, the fermented product of the present invention has better bioavailability and is conducive to the comprehensive utilization of ginseng.

Claims

1. A ginseng fermentation product, characterized in that, The ginseng fermentation product contains rare ginsenosides S-RG3 (0.02-0.1 mg / mL), R-Rg3 (0.009-0.029 mg / mL), Rg5 (0.087-0.245 mg / mL), CK (0.013-0.044 mg / mL), Rg2 (0.14-0.45 mg / mL), F2 (0.028-0.032 mg / mL), F4 (0.066-0.085 mg / mL), Rh2 (0.007-0.015 mg / mL), Rh4 (0.042-0.055 mg / mL), Rk2 (0.002-0.004 mg / mL), Rh3 (0.001-0.002 mg / mL), and PPT (0.019-0.026 mg / mL). The ginseng ferment is prepared by a stepwise mixed fermentation of two edible fungi, yeast and lactic acid bacteria, wherein the preparation method includes the following steps: Step (1): Mix the raw materials to be fermented; Step (2): Inoculate the mixture obtained in step (1) with yeast for fermentation; the weight ratio of the yeast to the mixture is (0.5~10):100; Step (3): Sterilization; Step (4): Inoculate with lactic acid bacteria for fermentation. The weight ratio of lactobacillus to the mixture is (0.5~10):

100. Step (5): Filter to obtain fermentation filtrate; Step (6): Sterilization.

2. The ginseng fermentation product according to claim 1, characterized in that, The ginseng fermentation product contains rare ginsenosides S-RG3 (0.04-0.060 mg / mL), R-Rg3 (0.013-0.029 mg / mL), Rg5 (0.129-0.241 mg / mL), CK (0.018-0.044 mg / mL), Rg2 (0.14-0.45 mg / mL), F2 (0.028-0.032 mg / mL), F4 (0.066-0.085 mg / mL), Rh2 (0.007-0.015 mg / mL), Rh4 (0.042-0.055 mg / mL), Rk2 (0.002-0.004 mg / mL), Rh3 (0.001-0.002 mg / mL), and PPT (0.019-0.026 mg / mL).

3. The ginseng fermentation product according to claim 1 or 2, characterized in that, The Rg2 is S-Rg2 and / or R-Rg2; the Rh2 is S-Rh2 and / or R-Rh2.

4. A method for preparing the ginseng ferment product according to any one of claims 1-3, comprising the following steps: Step (1): Mix the raw materials to be fermented; Step (2): Inoculate the mixture obtained in step (1) with yeast for fermentation; the weight ratio of the yeast to the mixture is (0.5~10):100; Step (3): Sterilization; Step (4): Inoculate with lactic acid bacteria for fermentation. The weight ratio of lactobacillus to the mixture is (0.5~10):

100. Step (5): Filter to obtain fermentation filtrate; Step (6): Sterilization.

5. The method for preparing ginseng fermentation product according to claim 4, wherein, In step (2), the weight ratio of the yeast to the mixture is (5~10):100; in step (4), the weight ratio of the lactobacillus to the mixture is (0.5~5):

100.

6. The preparation method according to claim 4, wherein, The raw materials to be fermented include ginseng, a carbon source, and a nitrogen source.

7. The preparation method according to claim 5, wherein, The raw materials to be fermented include ginseng, a carbon source, and a nitrogen source.

8. The preparation method according to claim 6, wherein, The ginseng is selected from ginseng root, ginseng flower, ginseng berry, ginseng stem and leaf and / or ginseng extract; the carbon source is selected from glucose, fructose, sucrose and / or maltose; and the nitrogen source is selected from ammonium sulfate, ammonium chloride, casein and / or milk powder.

9. The preparation method according to claim 8, wherein, The ginseng extract was obtained by water extraction of ginseng.

10. The preparation method according to claim 9, wherein, The water extraction temperature is 80-100℃.

11. The preparation method according to claim 10, wherein, The water extraction temperature is 80~85℃.

12. The preparation method according to any one of claims 4-11, wherein, The yeast is selected from Saccharomyces cerevisiae, Pichia pastoris and / or Candida albicans; the lactic acid bacteria are selected from Lactobacillus pentosus, Bifidobacterium longum, Bifidobacterium breve, Lactobacillus acidophilus, Lactobacillus plantarum and / or Lactobacillus rhamnosus.

13. The preparation method according to any one of claims 4-11, wherein, Yeast fermentation is carried out using a shaker culture, while lactic acid bacteria fermentation is carried out using anaerobic or aerobic culture.

14. The preparation method according to any one of claims 4-11, wherein, Yeast fermentation temperature is 25~35℃, and lactic acid bacteria fermentation temperature is 30~37℃.

15. The preparation method according to claim 12, wherein, Yeast fermentation temperature is 25~35℃, and lactic acid bacteria fermentation temperature is 30~37℃.

16. The preparation method according to claim 13, wherein, Yeast fermentation temperature is 25~35℃, and lactic acid bacteria fermentation temperature is 30~37℃.

17. The preparation method according to claim 14, wherein, Yeast fermentation temperature is 28~30℃, and lactic acid bacteria fermentation temperature is 35~37℃.

18. The preparation method according to any one of claims 4-11, wherein, Yeast fermentation time is 48-240 hours; lactic acid bacteria fermentation time is 48-240 hours.

19. The preparation method according to claim 12, wherein, Yeast fermentation time is 48-240 hours; lactic acid bacteria fermentation time is 48-240 hours.

20. The preparation method according to claim 13, wherein, Yeast fermentation time is 48-240 hours; lactic acid bacteria fermentation time is 48-240 hours.

21. The preparation method according to claim 14, wherein, Yeast fermentation time is 48-240 hours; lactic acid bacteria fermentation time is 48-240 hours.

22. The preparation method according to claim 18, wherein, Yeast fermentation time is 96-192 hours; lactic acid bacteria fermentation time is 96-240 hours.

23. The preparation method according to any one of claims 4-11, wherein, Step (3) includes adjusting the pH to 5.5-7.

5.

24. The preparation method according to claim 12, wherein, Step (3) includes adjusting the pH to 5.5-7.

5.

25. The preparation method according to claim 13, wherein, Step (3) includes adjusting the pH to 5.5-7.

5.

26. The preparation method according to claim 14, wherein, Step (3) includes adjusting the pH to 5.5-7.

5.

27. The preparation method according to claim 18, wherein, Step (3) includes adjusting the pH to 5.5-7.

5.

28. The preparation method according to claim 23, wherein, Step (3) includes adjusting the pH to 6-7.5 before lactic acid bacteria fermentation.

29. The ginseng fermentation product prepared by the preparation method according to any one of claims 5-28.

30. A pharmaceutical product, health food product, or skin care product containing the ginseng ferment as described in any one of claims 1-3 and 29.

31. A food product containing the ginseng fermentation product according to any one of claims 1-3 and 29.

32. The use of the ginseng ferment as described in any one of claims 1-3 and 29 in the preparation of medicines, health foods or skin care products that enhance immunity.

33. The use of the ginseng fermented product according to any one of claims 1-3 and 29 in the preparation of food products that enhance immunity.

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