Probiotics of fermented red ginseng and application of fermented red ginseng extract in hangover alleviating and liver protection

Abstract

The invention provides application of probiotics of fermented red ginseng and a fermented red ginseng extract to hangover alleviating and liver protection, and relates to the technical field of biological fermentation, the probiotics comprise plant lactobacillus KMHD-1 and / or lactobacillus gasseri KMBGI 03, data display shows that the probiotics have the content of the fermented red ginseng, and the fermented red ginseng extract has the content of the fermented red ginseng. The total amount of ginsenoside in the red ginseng extracting solution treated by the plant lactobacillus KMHD-1 or the lactobacillus gasseri KMBGI 03 is obviously increased; the ginsenoside content of the red ginseng extracting solution treated by the probiotics consisting of the plant lactobacillus KMHD-1 and the lactobacillus gasseri KMBGI 03 is higher than the ginsenoside content of the red ginseng extracting solution independently treated by the plant lactobacillus KMHD-1 or the lactobacillus gasseri KMBGI 03. The probiotics provided by the invention can increase the content of various ginsenosides at the same time. The technical problem that in the prior art, a probiotic capable of converting multiple rare ginsenosides at the same time is lacked is solved.

Description

Technical Field

[0001] The invention relates to the technical field of biological fermentation, and in particular to application of probiotics of fermented red ginseng and fermented red ginseng extract thereof in sobering up and protecting the liver. Background Art

[0002] Ginseng is the dried root and rhizome of the Araliaceae plant Panax ginseng. It is a traditional precious Chinese medicine and is known as the "king of herbs" and "panacea". Ginseng has certain requirements for the growth environment, and the requirements for the growth environment are relatively harsh. There are rotation restrictions on the planting of ginseng. Even in the case of transplanting, the land needs to be cultivated for 3-4 years, and generally it needs to be cultivated for about 7 years. Therefore, how to make good use of ginseng resources is a hot research direction. Ginseng saponins are the main active ingredients of ginseng, but the main saponins in ginseng have high content but low activity, and the bioavailability is about 2%. Most ginsenosides are difficult to be directly absorbed after oral administration. Some saponins are converted into rare ginsenosides through metabolism by intestinal microorganisms before they can enter the blood to exert biological activity. Therefore, using microorganisms to convert the main saponins into rare ginsenosides to improve bioavailability is an important research topic at present.

[0003] Red ginseng is a cooked product of ginseng, which is prepared through soaking, washing, steaming, airing and drying. The content of rare ginsenosides Rg3, Rg5 and Rk1 in processed red ginseng is significantly higher than that in raw sun-dried ginseng (i.e. original ginseng). Traditional red ginseng products are mainly red ginseng taproots, and red ginseng whiskers are by-products of red ginseng products, and the content of ginsenosides in red ginseng whiskers is higher than that in taproots. Taking red ginseng whiskers as the research object can enhance the overall utilization value of red ginseng. There are currently few public technologies for the method of fermenting and transforming a variety of rare ginsenosides with bacteria that can be used in food.

[0004] In view of this, the present invention is proposed. Summary of the invention

[0005] One of the purposes of the present invention is to provide a probiotic for fermenting red ginseng, so as to at least solve the technical problem in the prior art that there is a lack of a probiotic that can simultaneously transform multiple rare ginsenosides.

[0006] The second object of the present invention is to provide the use of the above-mentioned probiotics in increasing the rare ginsenoside content of red ginseng extract, preparing a product for increasing the rare ginsenoside content of red ginseng extract, or preparing a fermented red ginseng extract.

[0007] The third object of the present invention is to provide a method for increasing the content of rare ginsenosides in red ginseng extract.

[0008] A fourth object of the present invention is to provide a fermentation agent for increasing the rare ginsenoside content in red ginseng liquid.

[0009] A fifth object of the present invention is to provide a method for preparing a fermented red ginseng extract.

[0010] A sixth object of the present invention is to provide a fermented red ginseng extract.

[0011] The seventh object of the present invention is to provide the use of the above-mentioned fermented red ginseng extract in the preparation of a product for sobering up and protecting the liver.

[0012] In order to achieve the above-mentioned purpose of the present invention, the following technical solutions are particularly adopted: In a first aspect, the present invention provides a probiotic for fermenting red ginseng, wherein the probiotic comprises Lactobacillus plantarum KMHD-1 and / or Lactobacillus gasseri KMBGI 03; The Lactobacillus plantarum KMHD-1 ( Lactiplantibacillus plantarum The deposit number of KMHD-1 in the CGMCC is CGMCC No.28269; The Lactobacillus gasseri KMBGI 03 ( Lactobacillus gasseri The deposit number of KMBGI 03 in the CGMCC is CGMCC No.17177.

[0013] In a second aspect, the present invention provides the use of the above-mentioned probiotics in any of the following: A-1. Increase the content of rare ginsenosides in red ginseng extract; A-2. Preparation of a product for increasing the content of rare ginsenosides in red ginseng extract; A-3. Prepare fermented red ginseng extract.

[0014] In a third aspect, the present invention provides a method for increasing the rare ginsenoside content of a red ginseng extract, comprising fermenting the red ginseng extract using the probiotics described in claim 1.

[0015] Further, the method includes adding probiotic liquid to red ginseng extract for fermentation; The fermentation conditions are static culture at 30-42°C for 24-96h; After the fermentation, the fermentation step also includes a 90-100° C. water bath for 3 hours; The volume ratio of the probiotic solution to the red ginseng extract is 0.03-0.1:1.

[0016] Furthermore, the probiotic liquid includes Lactobacillus plantarum KMHD-1 ( Lactiplantibacillus plantarum KMHD-1) and / or Lactobacillus gasseri KMBGI 03 ( Lactobacillus gasseri KMBGI 03) bacterial solution; The Lactobacillus plantarum KMHD-1 ( Lactiplantibacillus plantarumKMHD-1) bacterial suspension OD 600 The value is 2.0~2.2; The Lactobacillus gasseri KMBGI 03 ( Lactobacillus gasseri KMBGI 03) OD of bacterial culture 600 The value is 1.2~1.4.

[0017] Furthermore, the probiotic liquid includes Lactobacillus plantarum KMHD-1 ( Lactiplantibacillus plantarum KMHD-1) and Lactobacillus gasseri KMBGI 03 ( Lactobacillus gasseri KMBGI 03) bacterial liquid, the Lactobacillus plantarum KMHD-1 ( Lactiplantibacillus plantarum KMHD-1) and Lactobacillus gasseri KMBGI 03 ( Lactobacillus gasseri KMBGI 03) The ratio of bacterial liquid is 0.8~1.5:1.

[0018] In a fourth aspect, the present invention provides a fermentation agent for increasing the rare ginsenoside content in red ginseng liquid, comprising the above-mentioned probiotics.

[0019] In a fifth aspect, the present invention provides a method for preparing a fermented red ginseng extract, comprising taking the fermented red ginseng extract obtained by fermentation with the above-mentioned probiotics, or the fermented red ginseng extract obtained by fermentation with the above-mentioned starter, or the fermented red ginseng extract prepared by the above-mentioned method, centrifuging to obtain the supernatant, and freeze-drying to obtain the fermented red ginseng extract.

[0020] In a sixth aspect, the present invention provides a fermented red ginseng extract prepared by the above-mentioned preparation method.

[0021] In a seventh aspect, the present invention provides the use of the above-mentioned fermented red ginseng extract in the preparation of a product for sobering up and protecting the liver.

[0022] The present invention provides a probiotic for fermenting red ginseng. Both Lactobacillus plantarum KMHD-1 and Lactobacillus gasseri KMBGI 03 can increase the content of rare ginsenosides in red ginseng extract. Data show that the total amount of ginsenosides in the red ginseng extract treated with Lactobacillus plantarum KMHD-1 or Lactobacillus gasseri KMBGI 03 is significantly increased, wherein the contents of Rh1, 20(R)-Rg2, 20(S)-Rg3, 20(R)-Rg3, Rk1 and Rg5 are increased, and the ginsenoside content of the red ginseng extract treated with the probiotics composed of Lactobacillus plantarum KMHD-1 and Lactobacillus gasseri KMBGI 03 is higher than the ginsenoside content of the red ginseng extract treated with Lactobacillus plantarum KMHD-1 or Lactobacillus gasseri KMBGI 03 alone. The probiotic provided by the present invention can increase the content of multiple ginsenosides at the same time. The technical problem of the lack of a probiotic that can simultaneously convert multiple rare ginsenosides in the prior art is solved. BRIEF DESCRIPTION OF THE DRAWINGS

[0023] In order to more clearly illustrate the specific implementation methods of the present invention or the technical solutions in the prior art, the drawings required for use in the specific implementation methods or the description of the prior art will be briefly introduced below. Obviously, the drawings described below are some implementation methods of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying creative work.

[0024] Figure 1 The biochemical index ALT test results of the fermented red ginseng extract provided in Experiment 2 of the present invention on rats with acute alcoholic liver injury; Figure 2 The test results of the biochemical index AST on rats with acute alcoholic liver injury by the fermented red ginseng extract provided in Experiment 2 of the present invention; Figure 3 The biochemical index GSH test results of the fermented red ginseng extract provided in Experiment 2 of the present invention on rats with acute alcoholic liver injury; Figure 4 The biochemical index MDA test results of the fermented red ginseng extract provided in Experiment 2 of the present invention on rats with acute alcoholic liver injury; Figure 5 The biochemical index TG test results of the fermented red ginseng extract provided in Experiment 2 of the present invention on rats with acute alcoholic liver injury; Figure 6 This is an H&E staining image of the liver tissue of rats with acute alcoholic liver injury treated with the fermented red ginseng extract provided in Experiment 2 of the present invention. DETAILED DESCRIPTION

[0025] Unless otherwise defined herein, scientific and technical terms used in conjunction with the present invention shall have the meanings commonly understood by those of ordinary skill in the art. The meaning and scope of the terms should be clear, however, in the case of any potential ambiguity, the definitions provided herein take precedence over any dictionary or external definitions. In this application, unless otherwise stated, the use of "or" means "and / or". In addition, the use of the term "including" and other forms is non-limiting.

[0026] The methods and techniques of the present invention are generally performed according to conventional methods well known in the art and as described in various general and more specific references that are cited and discussed throughout the present specification unless otherwise indicated.

[0027] In one aspect, the present invention provides a probiotic for fermenting red ginseng, wherein the probiotic comprises Lactobacillus plantarum KMHD-1 and / or Lactobacillus gasseri KMBGI 03; The Lactobacillus plantarum KMHD-1 ( Lactiplantibacillus plantarum The deposit number of KMHD-1 in the CGMCC is CGMCC No.28269; The Lactobacillus gasseri KMBGI 03 ( Lactobacillus gasseri The deposit number of KMBGI 03 in the CGMCC is CGMCC No.17177.

[0028] Storage Instructions of Lactobacillus plantarum KMHD-1 Bacterial strain name: Lactobacillus plantarum KMHD-1; Latin name: Lactiplantibacillus plantarum ; Depository: General Microbiology Center, China Microbiological Culture Collection Administration; Abbreviation of depository institution: CGMCC; Address: No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing; Deposit date: August 28, 2023; Collection Center Collection Number: CGMCC No.28269.

[0029] Storage Instructions of Lactobacillus gasseri KMBGI 03 Bacterial species name: Lactobacillus gasseri KMBGI 03; Latin name: Lactobacillus gasseri ; Depository: General Microbiology Center, China Microbiological Culture Collection Administration; Address: No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing; Deposit date: January 14, 2019; Abbreviation of depository institution: CGMCC; Collection Center Collection Number: CGMCC No.17177.

[0030] Both Lactobacillus plantarum KMHD-1 and Lactobacillus gasseri KMBGI 03 can increase the content of rare ginsenosides in red ginseng extract. Data show that the total amount of ginsenosides in the red ginseng extract treated with Lactobacillus plantarum KMHD-1 or Lactobacillus gasseri KMBGI 03 is significantly increased, wherein the contents of Rh1, 20(R)-Rg2, 20(S)-Rg3, 20(R)-Rg3, Rk1 and Rg5 are increased, and the ginsenoside content of the red ginseng extract treated with the probiotics composed of Lactobacillus plantarum KMHD-1 and Lactobacillus gasseri KMBGI 03 is higher than the ginsenoside content of the red ginseng extract treated with Lactobacillus plantarum KMHD-1 or Lactobacillus gasseri KMBGI 03 alone. The probiotics provided by the present invention can increase the contents of multiple ginsenosides at the same time. The technical problem of the lack of a probiotic that can simultaneously convert multiple rare ginsenosides in the prior art is solved.

[0031] According to another aspect of the present invention, there is also provided the use of the above-mentioned probiotics in any of the following items: A-1. Increase the content of rare ginsenosides in red ginseng extract; A-2. Preparation of a product for increasing the content of rare ginsenosides in red ginseng extract; A-3. Prepare fermented red ginseng extract.

[0032] Among them, the rare ginsenosides include the total amount of rare ginsenosides, or at least one of Rh1, 20(R)-Rg2, 20(S)-Rg3, 20(R)-Rg3, Rk1 and Rg5.

[0033] According to another aspect of the present invention, there is also provided a method for increasing the rare ginsenoside content of a red ginseng extract, comprising fermenting the red ginseng extract with the above-mentioned probiotics.

[0034] The above-mentioned probiotics can transform a variety of rare ginsenosides during the fermentation of red ginseng extract to increase the total amount of ginsenosides in the red ginseng extract.

[0035] In some specific embodiments, the method comprises adding a probiotic solution to a red ginseng extract for fermentation.

[0036] In some specific embodiments, the fermentation condition is static culture at 30-42° C. for 24-96 hours, preferably static culture at 37° C. for 72 hours.

[0037] In some specific embodiments, the fermentation further comprises a 90-100° C. water bath for 3 hours.

[0038] In some specific embodiments, the volume ratio of the probiotic solution to the red ginseng extract is 0.03-0.1:1.

[0039] The probiotic solution is added to the red ginseng extract at a ratio of 3% to 10%, and the fermentation effect is best at a ratio of 5%. The volume ratio of the probiotic solution to the red ginseng extract may be, but is not limited to, 0.03:1, 0.04:1, 0.05:1, 0.06:1, 0.07:1, 0.08:1, 0.09:1 or 0.1:1, or any ratio between 0.03 and 0.1:1, preferably 0.05:1.

[0040] In some specific embodiments, the probiotic solution includes Lactobacillus plantarum KMHD-1 ( Lactiplantibacillus plantarum KMHD-1) and / or Lactobacillus gasseri KMBGI 03 ( Lactobacillus gasseri KMBGI 03) bacterial solution; The Lactobacillus plantarum KMHD-1 ( Lactiplantibacillus plantarum KMHD-1) bacterial suspension OD 600 The value is 2.0~2.2; The Lactobacillus gasseri KMBGI 03 ( Lactobacillus gasseri KMBGI 03) OD of bacterial culture 600 The value is 1.2~1.4.

[0041] Among them, the bacterial liquid is the fermentation seed liquid. After the glycerol tube of the bacterial strain is thawed, the bacterial liquid is separated by drawing lines on a plate on an ultra-clean bench to obtain a single colony, and the single colony is inoculated into MRS liquid culture medium and statically cultured to obtain the fermentation seed liquid of the strain.

[0042] In some specific embodiments, the probiotic solution includes Lactobacillus plantarum KMHD-1 ( Lactiplantibacillus plantarum KMHD-1) and Lactobacillus gasseri KMBGI 03 ( Lactobacillus gasseri KMBGI 03) bacterial liquid, the Lactobacillus plantarum KMHD-1 ( Lactiplantibacillus plantarum KMHD-1) and Lactobacillus gasseri KMBGI 03 ( Lactobacillus gasseri KMBGI 03) The ratio of bacterial liquid is 0.8~1.5:1.

[0043] According to another aspect of the present invention, there is also provided a fermentation agent for increasing the rare ginsenoside content of red ginseng extract, comprising the above-mentioned probiotics.

[0044] According to another aspect of the present invention, a method for preparing a fermented red ginseng extract is also provided, comprising taking the fermented red ginseng extract obtained by fermentation with the above-mentioned probiotics, or the fermented red ginseng extract obtained by fermentation with the above-mentioned starter, or the fermented red ginseng extract prepared by the above-mentioned method, centrifuging to obtain the supernatant, and freeze-drying to obtain the fermented red ginseng extract.

[0045] According to another aspect of the present invention, a fermented red ginseng extract is provided, which is prepared by the above-mentioned preparation method.

[0046] According to another aspect of the present invention, there is also provided a use of the fermented red ginseng extract in the preparation of a product for sobering up and protecting the liver.

[0047] The technical solution of the present invention will be clearly and completely described below in conjunction with the embodiments. Obviously, the described embodiments are part of the embodiments of the present invention, rather than all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0048] Red ginseng extract: Weigh red ginseng and add 10 times of pure water, extract at 90-100°C for 2h, filter to obtain a first extract, add 10 times of water to the residue, extract at 90-100°C for 2h, filter to obtain a second extract; mix the first extract and the second extract and concentrate under reduced pressure by 2 times to obtain a red ginseng extract for use in subsequent examples.

[0049] Preparation of fermentation seed solution: After thawing the glycerol tubes of the two bacterial strains, separate the bacterial solution by drawing lines on a plate on an ultra-clean bench to obtain single colonies, inoculate the single colonies into MRS liquid culture medium, and culture them statically to obtain the fermentation seed solution of the strains.

[0050] Red ginseng powder 1: Use traditional water extraction method, extract at 100°C for 2h, extract once, and dry the extract to obtain red ginseng powder.

[0051] Red ginseng powder 2: prepared by traditional water extraction plus enzymatic treatment, specifically comprising: extracting red ginseng twice at 100°C, cooling to 45°C, adding the extract into an enzymatic hydrolysis tank, adding 3g / L of cellulase and pectinase respectively, enzymatic hydrolysis at 45°C for 3h, performing enzyme inactivation treatment at 95°C for 10min, cooling to room temperature and spray drying to obtain red ginseng powder.

[0052] Example 1 Isolation, identification and preservation of strains 1. Isolation and purification of Lactobacillus gasseri KMBGI 03 strain Weigh 0.2g of healthy stool sample, add 1.8mL PBS or saline, place in a metal bath at 300rpm, shake for 10min; centrifuge at 50g for 15min, take 1mL of upper suspension; centrifuge at 8000g for 5min, remove the upper liquid, add 1mL PBS to the precipitate and resuspend; dilute the bacterial suspension 10 times, 100 times, and 1000 times; dip a cotton swab in the bacterial suspension for plate coating, and take 10-3, 10-4 dilutions from fresh feces for coating; invert the plate and place in an anaerobic incubator at 37℃ for 48h. Pick the single colony that turns the bromocresol green medium yellow, streak it again on the MRS medium, and culture it anaerobically at 37℃. The obtained colony is streaked and purified multiple times to obtain a single bacterium, named KMBGI 03 strain.

[0053] 2. Isolation and purification of Lactobacillus plantarum KMHD-1 strain Take 2.5 g of sample from sauerkraut, add it to a sterile bag containing 22.5 mL of sterile saline, pat it for 2 minutes, prepare MRS medium containing bromocresol green, obtain single colonies by 10-fold gradient dilution method, culture it anaerobically at 37°C for 36-48 h, pick out the single colony that makes the bromocresol green medium turn yellow, streak it again on MRS medium, culture it anaerobically at 37°C, and purify the obtained colonies by multiple streaking to obtain a single bacterium, which was named KMHD-1 strain.

[0054] 3. Identification of strains (1) Morphological identification: observe the morphology of single bacterial colonies after multiple purifications: The KMBGI 03 strain is round, white or off-white, translucent, smooth and convex with neat edges. It is purple in Gram staining, negative in enzyme contact reaction, and rod-shaped in microscopic examination.

[0055] The KMHD-1 strain is round, white or off-white, 1-2 μm in diameter, with a smooth and moist surface, an arc-shaped protrusion in the middle, opaque, and neat edges without halos; Gram staining is purple, enzyme contact reaction is negative, and microscopic examination shows rod-shaped.

[0056] (2) 16S identification: Using colony DNA as a template, the universal primers 27F and 1492R for bacterial sequencing were used to amplify the 16SrRNA gene fragment. The PCR amplification reaction system and amplification procedure are shown in Tables 1 and 2. The amplicon sequences were aligned, and the PCR products were sent to the company for 16SrRNA gene amplicon sequencing.

[0057] 27F: 5'-AGAGTTTGATCCTGGCTCAG-3' (SEQ ID NO. 1); 1492R: 5'-TACGGCTACCTTGTTACGACTT-3' (SEQ ID NO. 2).

[0058] Table 1 PCR reaction system

[0059] Table 1 PCR reaction procedure

[0060] 16S sequence of KMBGI 03 strain:

[0061] 16S sequence of KMHD-1 strain:

[0062] The base sequences in the determination results were input into the NCBI (http: / / blast.ncbi.nlm.nih.gov / Blast.cgi) database for comparison, and the KMBGI 03 strain was identified as Lactobacillus gasseri and the KMHD-1 strain was identified as Lactobacillus plantarum. Lactobacillus gasseri KMBGI 03) and Lactobacillus plantarum KMHD-1 ( Lactobacillus gasseri KMBGI 03) and deposited in the General Microbiology Center of China Microbiological Culture Collection Administration.

[0063] Example 2 Experiment on strain resistance to artificial gastric acid / intestinal fluid The gastric acid / intestinal fluid resistance of the strain obtained in Example 1 was tested according to the following specific operations.

[0064] Artificial gastric acid resistance test: weigh 2.0g NaCl + 3.2g pepsin, add 7.0ml HCl (concentrated), dilute to 1000ml with ddH2O, mix well; take 50ml of the solution, adjust the pH to 2.0 and 3.0, filter and sterilize for later use; activate the glycerol seed twice, take 1ml of the overnight cultured bacterial solution in a 1.5ml centrifuge tube, centrifuge (5000 rpm for 5min) and remove the supernatant; resuspend the bacteria with an equal volume of simulated gastric fluid, mix well and immediately aspirate 100μl of the above bacterial solution, mix with 90 0μl sterile physiological saline, and make gradient dilutions, take 10-6 and 10-7 for coating at 0h; the sampled bacterial solution is placed in a 37℃ incubator for incubation, and samples are taken at the time point of 2h to simulate gastric fluid according to the original experimental time length for dilution and coating; at each sampling time point, the bacterial solution is taken out from the centrifuge tube, mixed and sampled for serial dilution; two parallel samples are made for each strain, and each parallel sample is repeated 2 times; after the coated plate is placed in a 37℃ incubator for incubation for 36~48h, the colony count is performed and the data is recorded; Artificial intestinal fluid resistance experiment: weigh 6.8g potassium dihydrogen phosphate ddH2O to 250ml; measure 77ml 0.2M NaOH, ddH2O to 500ml; mix 250ml potassium dihydrogen phosphate with 500ml NaOH solution, add 10.0g pancreatic enzyme and 0.3% bile salt, adjust pH to 6.8±0, make up to 1000ml, filter and sterilize for use; activate glycerol seed twice, take 1ml overnight cultured bacterial solution in a 1.5ml centrifuge tube, centrifuge at 5000 rpm for 5min to remove supernatant; resuspend the bacteria with an equal volume of simulated intestinal fluid, immediately aspirate 100μl of the above bacterial solution after mixing, mix it into 900μl sterile saline, and make gradient Dilution, take 10-6 and 10-7 at 0h for coating; place the sampled bacterial solution in a 37℃ incubator for culture, and take samples at 3 and 6h of simulated intestinal fluid according to the original length of the experiment for dilution and coating; at each sampling time point, take out the bacterial solution from the centrifuge tube, mix it and take samples for serial dilution; make two parallel samples for each strain, and repeat 2 times for each parallel sample; after the coated plate is placed in a 37℃ incubator for culture for 36~48h, count the colonies and record the data as shown in Table 3.

[0065] Table 3 Experimental data of strains simulating gastric acid and intestinal fluid

[0066] After the two strains were treated with simulated gastric acid and simulated intestinal fluid, the number of viable bacteria exceeded 1×10 6 CFU / mL, with good tolerance to gastric and intestinal juices.

[0067] Example 3 A method for increasing the rare ginsenoside content of a red ginseng extract, comprising the steps of: pasteurizing the red ginseng extract for 30 minutes, and then 600 The fermented seed liquid of Lactobacillus gasseri KMBGI 03 with a value of 1.3 was added to the red ginseng extract at a ratio of 5% (V / V), and the mixture was allowed to ferment at 37°C for 72 hours. After fermentation, the mixture was placed in a water bath at 90-100°C for 3 hours to obtain the fermented red ginseng extract.

[0068] Example 4 A method for increasing the rare ginsenoside content of a red ginseng extract, comprising the steps of: pasteurizing the red ginseng extract for 30 minutes, and then 600 The fermented seed liquid of Lactobacillus plantarum KMHD-1 with a value of 2.1 was added to the red ginseng extract at a ratio of 5% (V / V), and fermented at 37°C for 72 hours. After fermentation, it was water bathed at 90-100°C for 3 hours to obtain the fermented red ginseng extract.

[0069] Example 5 A method for increasing the rare ginsenoside content of a red ginseng extract, comprising the steps of: pasteurizing the red ginseng extract for 30 minutes, and then 600 The fermentation seed liquid of Lactobacillus gasseri KMBGI 03 with an OD value of 1.3 600 The fermented seed liquid of Lactobacillus plantarum KMHD-1 with a value of 2.1 was mixed in a ratio of 1:1, and then added to the red ginseng extract at a ratio of 5% (V / V), and fermented at 37°C for 72 hours. After fermentation, it was water bathed at 90-100°C for 3 hours to obtain the fermented red ginseng extract.

[0070] Example 6 The difference from Example 5 is that OD 600 The fermentation seed liquid of Lactobacillus gasseri KMBGI 03 with an OD value of 1.3 600 The fermented seed liquid of Lactobacillus plantarum KMHD-1 with a value of 2.1 was mixed in a ratio of 1:1 and then added to the red ginseng extract at a ratio of 3% (V / V). Example 7 The difference from Example 5 is that OD 600 The fermentation seed liquid of Lactobacillus gasseri KMBGI 03 with an OD value of 1.3 600 The fermented seed liquid of Lactobacillus plantarum KMHD-1 with a value of 2.1 was mixed in a ratio of 1:1 and then added to the red ginseng extract at a ratio of 10% (V / V).

[0071] Example 8 Different from Example 5, the fermentation was allowed to stand at 30° C. for 72 h, and after fermentation, the mixture was water bathed at 90-100° C. for 3 h to obtain the fermented red ginseng extract.

[0072] Example 9 Different from Example 5, the fermentation was allowed to stand at 42° C. for 72 h, and after fermentation, the mixture was water bathed at 90-100° C. for 3 h to obtain the fermented red ginseng extract.

[0073] Example 10 Different from Example 5, the fermentation was allowed to stand at 37° C. for 24 h, and after fermentation, the mixture was water bathed at 90-100° C. for 3 h to obtain the fermented red ginseng extract.

[0074] Embodiment 11 Different from Example 5, the fermentation was allowed to stand at 37° C. for 48 h, and after fermentation, the mixture was water bathed at 90-100° C. for 3 h to obtain the fermented red ginseng extract.

[0075] Example 12 Different from Example 5, the fermentation was allowed to stand at 37° C. for 96 h, and after fermentation, the mixture was water bathed at 90-100° C. for 3 h to obtain the fermented red ginseng extract.

[0076] Embodiment 13 The difference from Example 5 is that the fermentation was not followed by a 3 h water bath at 90-100°C.

[0077] Comparative Example 1 The difference from Example 3 is that OD 600 The value is 1.3 and the fermented seed liquid of Lactobacillus gasseri LG-G12 (preservation number: CCTCCNO: M2019829) is used to ferment the red ginseng extract.

[0078] Comparative Example 2 The difference from Example 4 is that OD 600 The value is 2.1 and the fermented seed liquid of Lactobacillus plantarum JYLP-326 (preservation number: CGMCC NO.18038) is fermented into red ginseng extract.

[0079] Comparative Example 3 The difference from Example 5 is that OD 600 The fermentation seed liquid of Lactobacillus gasseri LG-G12 with an OD value of 1.3 was used instead of 600 The fermentation seed liquid of Lactobacillus gasseri KMBGI 03 had a value of 1.3.

[0080] Comparative Example 4 The difference from Example 5 is that OD 600 The fermented seed liquid of Lactobacillus plantarum JYLP-326 with an OD value of 2.1 was used instead of 600 The fermentation seed liquid of Lactobacillus plantarum KMHD-1 had a value of 2.1.

[0081] Embodiments 14 to 24 A fermented red ginseng extract is prepared by centrifuging and filtering the fermented red ginseng extracts prepared in Examples 3 to 13, respectively, to obtain red ginseng fermented broths, freeze-drying, and collecting red ginseng fermented extracts.

[0082] Comparative Examples 5 to 8 The difference from Example 14 is that the fermented red ginseng extracts prepared in Comparative Examples 1 to 4 are used.

[0083] Experiment 1 Detection of rare ginsenoside content in red ginseng fermented extract Detection method: Ultra high performance liquid chromatography was used, with acetonitrile containing 0.1% formic acid as mobile phase B and water containing 0.1% formic acid as mobile phase C, for gradient elution; the chromatographic column was ACQUITY UPLC BEH Shield RP18 (2.1mm×100mm, 1.7μm); the flow rate was 0.4 mL / min; the column temperature was 30℃; the injection volume was 3μL; and the autosampler temperature was 4℃.

[0084] UPLC condition optimization: According to the spectrum, the proportion of mobile phase at different times was adjusted, and the influence of different proportions on separation and column efficiency was analyzed. After multiple adjustments and optimizations, the gradient elution program was as follows: 0-5 min, 20-30% B; 5-12 min, 30-33% B; 12-13 min, 33-40% B; 13-23 min, 40-47% B; 23-24 min, 47-60% B; 24-26 min, 60-65% B; 26-33 min, 65-78% B; 33-35 min, 78-80% B; 35-36 min, 80-20% B; 36-41 min, 20% B. The test results are shown in Table 4.

[0085] Table 4 Comparison of rare ginsenoside contents

[0086] As shown in Table 4, the content and total amount of each ginsenoside of Examples 14 to 24 are higher than those of Comparative Examples 5 to 8, indicating that the probiotics provided by the present application can promote the conversion of rare ginsenosides and increase the content of rare saponins in the red ginseng extract. Compared with Example 14, the ginsenoside content of Comparative Example 5 is lower, indicating that the Lactobacillus gasseri KMBGI 03 provided by the present application is better than the Lactobacillus gasseri in Comparative Example 5; compared with Example 15, the ginsenoside content of Comparative Example 6 is lower, indicating that the Lactobacillus plantarum KMHD-1 provided by the present application is better than the Lactobacillus plantarum in Comparative Example 6.

[0087] The total amount of ginsenosides in Examples 16 to 18 is higher than that in Examples 14 and 15, indicating that the combined use of Lactobacillus gasseri and Lactobacillus plantarum can further promote the conversion of rare ginsenosides. Compared with Example 16, the ginsenoside content of Comparative Examples 7 and 8 is reduced, indicating that not any combination of Lactobacillus gasseri and Lactobacillus plantarum can achieve the effect of increasing the ginsenoside content in Example 16. The total amount of ginsenosides in Example 18 is the highest, with a total of 20 (S) -Rg3 and 20 (R) -Rg3 of 32.38 mg / g, and the total content of 20 (S) -Rg3, 20 (R) -Rg3, Rk1 and Rg5 is more than 20 times that of red ginseng powder 1, which has a significant advantage over red ginseng powder 2.

[0088] Experiment 2: Alleviating effect of fermented red ginseng extract on acute alcoholic liver injury in rats Materials: 65° liquor (Beijing Red Star Co., Ltd.), distilled water, red ginseng extract freeze-dried powder (RG), red ginseng fermented freeze-dried powder (FRG).

[0089] Grouping: Wistar rats were divided into 4 groups, each with 5 blank groups, 8 model groups, 8 control groups RG, and 8 experimental groups FRG, for a total of 29 rats.

[0090] Experimental method: All animals were adaptively fed for one week, weighed and randomly divided into blank group, model group, control group RG (3600 mg / kg), and experimental group FRG (3600 mg / kg). There were 5 animals in the blank group and 8 animals in each of the other groups, and then they were numbered and recorded.

[0091] The dosage of the control group and the experimental group was 3600 mg / kg, and the gavage capacity was 10 mL / kg, that is, 360 mg dissolved in 1 mL of distilled water. Every morning, 10 mL / kg of body weight was gavaged: the blank group and the model group were gavaged with distilled water every day, the control group RG was treated with red ginseng extract prepared from unfermented red ginseng extract, and the experimental group was treated with fermented red ginseng extract prepared in Example 16, once a day for 14 days. Starting from the 8th day, except for the blank group, the model group, the red ginseng group and the fermented red ginseng group were gavaged with 10 mL / kg 65° liquor every day, and alcohol was gavaged once every 12 hours, for a total of 6 times, and the NC group was given an equal amount of distilled water. While gavaging alcohol, drug treatment was still given every day.

[0092] Serum collection: 12 hours after the last administration of alcohol, blood was collected from the abdominal aorta of the rats. After being kept at room temperature for 2 hours, the blood was centrifuged at 3500r / min for 15 minutes in a 4℃ low-temperature refrigerated centrifuge. The serum was collected and stored at -80℃ for subsequent biochemical index detection.

[0093] Liver tissue sampling: After the rats were killed, the liver tissue was quickly dissected and the total weight of the liver was recorded and photographed. The liver tissue of 1×1 cm was cut with scissors, and the liquid on the surface of the tissue was wiped off with gauze, and then placed on OCT for fusion for 5 minutes (on ice), and then frozen on dry ice. The subsequent sections were stained with H&E to observe the degree of fatty degeneration and inflammation of the liver tissue.

[0094] (1) Physiological and biochemical index detection: Changes in biochemical indexes in the blood. Biochemical indexes related to liver damage, such as ALT (alanine aminotransferase), AST (aspartate aminotransferase), TG (triglyceride), MDA (malondialdehyde) and GSH (glutathione), were measured using detection kits, and the differences among the groups were analyzed. Figure 1~Figure 5 shown.

[0095] Depend on Figure 1 It can be seen that the ALT content in serum of the model group was significantly higher than that of the blank group, indicating that the model was successfully established; compared with the model group, the ALT level of the experimental group was significantly lower, and the difference was statistically significant.

[0096] Depend on Figure 2 It can be seen that the AST content in serum of the model group was significantly higher than that of the blank group, indicating that the model was successful; compared with the model group, the AST level of the experimental group was significantly lower, and the difference was statistically significant.

[0097] Depend on Figure 3 It can be seen that the GSH content in serum of the model group was significantly lower than that of the blank group, indicating that the model was successful; the GSH level of the experimental group was significantly higher than that of the model group, and the difference was statistically significant.

[0098] Depend on Figure 4 It can be seen that the MDA content in serum of the model group was significantly higher than that of the blank group, indicating that the model was successful; the GSH level of the experimental group was significantly lower than that of the model group, and the difference was statistically significant.

[0099] Depend on Figure 5 It can be seen that the TG content in serum of the model group was significantly higher than that of the blank group, indicating that the model was successful; the TG level of the experimental group was significantly lower than that of the model group, and the difference was statistically significant.

[0100] (2) Changes in liver tissue such as Figure 6 As shown in the figure, the liver lobule structure of the rats in the blank group was clear and complete, the hepatic cords were arranged in an orderly manner, and there was no obvious lesion. In the model group, there were obvious dense fat droplets in the cytoplasm of the liver tissue of the rats, the hepatic lobule was destroyed, the structure was blurred, and bleeding spots and necrosis were visible. In the experimental group, a few fat droplets were seen in the liver tissue of the rats, the hepatic lobule was partially destroyed, the structure was relatively clear, and the liver damage was improved. The degree of improvement in the experimental group was better than that in the control group.

[0101] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention, rather than to limit it. Although the present invention has been described in detail with reference to the aforementioned embodiments, those skilled in the art should understand that they can still modify the technical solutions described in the aforementioned embodiments, or replace some or all of the technical features therein with equivalents. However, these modifications or replacements do not cause the essence of the corresponding technical solutions to deviate from the scope of the technical solutions of the embodiments of the present invention.

Claims

1. A probiotic for fermenting red ginseng, characterized in that: The probiotics include Lactobacillus plantarum KMHD-1 and / or Lactobacillus gasseri KMBGI 03; The Lactobacillus plantarum KMHD-1 ( Lactiplantibacillus plantarum KMHD-1) is deposited in the General Microbiology Center of China Microbiological Culture Collection Committee with the deposit number of CGMCC No.28269; The Lactobacillus gasseri KMBGI 03 ( Lactobacillus gasseri KMBGI 03) is deposited in the General Microbiology Center of China Microorganism Culture Collection Administration with the deposit number of CGMCC No.17177.

2. Use of the probiotics according to claim 1 in any of the following: A-1. Increase the content of rare ginsenosides in red ginseng extract; A-2. Preparation of a product for increasing the content of rare ginsenosides in red ginseng extract; A-3. Prepare fermented red ginseng extract.

3. A method for increasing the content of rare ginsenosides in red ginseng extract, characterized in that: The method comprises using the probiotic fermented red ginseng extract according to claim 1.

4. The method according to claim 3, characterized in that The method comprises adding probiotic liquid to red ginseng extract liquid for fermentation; The fermentation conditions are static culture at 30-42°C for 24-96h; After the fermentation, the fermentation step also includes a 90-100° C. water bath for 3 hours; The volume ratio of the probiotic solution to the red ginseng extract is 0.03-0.1:

1.

5. The method according to claim 4, characterized in that The probiotic liquid includes Lactobacillus plantarum KMHD-1 ( Lactiplantibacillus plantarum KMHD-1) and / or Lactobacillus gasseri KMBGI 03 ( Lactobacillus gasseri KMBGI 03) bacterial solution; The Lactobacillus plantarum KMHD-1 ( Lactiplantibacillus plantarum KMHD-1) bacterial culture OD 600 The value is 2.0~2.2; The Lactobacillus gasseri KMBGI 03 ( Lactobacillus gasseri KMBGI 03) OD of bacterial culture 600 The value is 1.2~1.

4.

6. The method according to claim 5, characterized in that The probiotic liquid includes Lactobacillus plantarum KMHD-1 ( Lactiplantibacillus plantarum KMHD-1) and Lactobacillus gasseri KMBGI 03 ( Lactobacillus gasseri KMBGI 03) bacterial liquid, the Lactobacillus plantarum KMHD-1 ( Lactiplantibacillus plantarum KMHD-1) and Lactobacillus gasseri KMBGI 03 ( Lactobacillus gasseri KMBGI 03) The ratio of bacterial liquid is 0.8~1.5:

1.

7. A fermentation agent for increasing the rare ginsenoside content of red ginseng extract, characterized in that: The invention comprises the probiotics according to claim 1.

8. A method for preparing a fermented red ginseng extract, characterized in that: The method comprises taking the fermented red ginseng extract obtained by fermentation with the probiotics according to claim 1, the fermented red ginseng extract obtained by fermentation with the starter according to claim 7, or the fermented red ginseng extract prepared by the methods according to claims 3 to 6, centrifuging to obtain the supernatant, and freeze-drying to obtain the fermented red ginseng extract.

9. A fermented red ginseng extract, characterized in that: Prepared by the preparation method according to claim 8.

10. Use of the fermented red ginseng extract according to claim 9 in the preparation of a product for sobering up and protecting the liver.

Images