Ginseng fermentation product, preparation method and application thereof

Abstract

This patent application discloses a ginseng fermentation product, its preparation method and application. In view of the problem that existing ginseng fermentation products have certain effects in whitening, anti-anemia, anti-inflammation, delaying cognitive impairment, and enhancing the body's immunity, but none of the effects are particularly clear and prominent, the ginseng fermentation product is prepared by fermenting the ginseng fermentation matrix with a specific probiotic composition. It is rich in rare ginsenosides, and the content of ginsenoside Rh4 with anti-fatigue effect is high, while the content of ginsenoside Rk1 and ginsenoside Rg5 with opposite effects on anti-fatigue is low. Experimental studies have shown that compared with existing ginseng fermentation products, the ginseng fermentation product of the present invention has a clearer and more prominent anti-fatigue effect. The preparation method of the ginseng fermentation product is simple and easy to industrialize. It can be widely used in major health fields such as functional foods and has broad application prospects.

Description

Technical Field

[0001] The present invention relates to the technical field of synthetic biology, and in particular to a ginseng fermentation product rich in specific rare ginsenosides, a biological fermentation preparation method thereof, and applications. Background Art

[0002] Ginseng (Panax ginseng CA ley.) is the dried root and rhizome of the Araliaceae plant Panax ginseng. It has the effects of tonifying vital energy, strengthening the body, promoting salivation, calming the mind, and improving intelligence. Ginsenosides are the main active ingredients of ginseng and are divided into natural ginsenosides and rare ginsenosides. Natural ginsenosides mainly include Rb1, Rb2, Rb3, Rc, Rg1, Re, Rf, etc., with a content generally ranging from 4-6%. Rare ginsenosides are generally metabolites of natural ginsenosides in the human body and include Rg3, Rk1, Rg5, CK, F2, Rh1, Rk3, Rh4, etc. Their natural content in ginseng is very low, only one in 100,000. Studies have shown that rare ginsenosides are significantly more effective than natural ginsenosides in anti-tumor, hypoglycemic, hypolipidemic, immune-enhancing, and antioxidant effects.

[0003] Natural ginsenosides can be absorbed and converted into rare ginsenosides in the body through intestinal microorganisms and enzymes. Ginsenosides are hydrolyzed in the acidic gastric fluid environment to produce deglycosylated metabolites. Glycoside hydrolases expressed by the intestinal flora (such as β-glucosidase, α-rhamnosidase, and xylosidase, etc.) then deglycosylate to produce secondary metabolites or aglycones (deglycosylated ginsenosides), namely rare ginsenosides. These are then absorbed into the systemic circulation through transmembrane transport and other means, thereby exerting their medicinal effects.

[0004] In related technical research, microbial fermentation of ginseng is considered one of the safest and most efficient methods for the in vitro conversion of ginsenosides. In fact, researchers have already used a single microorganism or a combination of microorganisms to ferment ginseng fermentation substrates, producing a variety of rare ginsenosides.

[0005] However, a technical problem with these existing technologies is that the biofermentation process produces a variety of ginsenoside compositions, which can include dozens of rare ginsenosides. These ginsenosides have varying biological effects, with some even antagonizing each other. Consequently, these ginsenoside products are more like a "jack of all trades," potentially offering benefits such as whitening, fighting anemia, combating inflammation, delaying cognitive impairment, and enhancing immunity, but without a single clear or prominent benefit. Summary of the Invention

[0006] In view of the above technical problems existing in the prior art, the object of the present invention is to provide a ginseng fermentation product rich in rare ginsenosides with enhanced anti-fatigue effect, a microbial fermentation preparation method and application thereof.

[0007] After literature research, the inventors discovered that among the rare ginsenosides, ginsenoside Rh4 is the most effective in combating fatigue, while ginsenosides Rk1 and Rg5 can inhibit neural excitation and promote sleep, effectively counteracting fatigue. Based on this, they concluded that increasing the content of ginsenoside Rh4 in ginseng fermentation products while simultaneously decreasing the content of ginsenosides Rk1 and Rg5 would allow the production of ginseng fermentation products with enhanced anti-fatigue properties.

[0008] However, the technical difficulty is that the use of microorganisms (probiotics) to transform natural ginsenosides to produce rare ginsenosides may be related to the transformation pathway. Among all rare ginsenosides, ginsenoside Rh4 is difficult to produce, while ginsenoside Rk1 and Rg5 are relatively easy to produce. Moreover, when a certain amount of ginsenoside Rh4 is produced, more ginsenoside Rk1 and Rg5 are often produced at the same time. This results in either a very low content of ginsenoside Rh4 in existing ginseng fermentation products, or a much higher content of ginsenoside Rk1 and Rg5 than that of ginsenoside Rh4, resulting in the ginseng fermentation product being unable to exhibit the anti-fatigue effect of ginsenoside Rh4 as a whole.

[0009] Furthermore, after in-depth research, the inventors found that fermenting the ginseng fermentation matrix with a specific composition of Lactobacillus plantarum, Lactobacillus paracasei, Lactobacillus casei, and Bifidobacterium adolescens can produce a ginseng fermentation product with a high content of ginsenoside Rh4 and a small difference in content from ginsenosides Rk1 and Rg5, so that the ginseng fermentation product exhibits better anti-fatigue effect, thereby completing the present invention.

[0010] The technical solution of the present invention includes:

[0011] 1. A ginseng fermentation product obtained by fermenting a ginseng fermentation substrate using a microbial strain, comprising ginsenoside Rh4, ginsenoside Rk1, and ginsenoside Rg5, which satisfies:

[0012] The content of ginsenoside Rh4 is ≥3% by weight; and

[0013] Ginsenoside Rh4 content - Ginsenoside Rk1 content - Ginsenoside Rg5 content ≥ - 5% by weight

[0014] The total amount of saponins in the ginseng fermentation product is taken as 100% by weight.

[0015] The ginseng fermentation product may be liquid or solid or a solid-liquid mixture, for example, fermented ginseng liquid or fermented ginseng powder.

[0016] 2. A bacterial composition comprising Lactobacillus plantarum, Lactobacillus casei, Lactobacillus paracasei and Bifidobacterium adolescens; wherein:

[0017] The Lactobacillus plantarum is Lactobacillus plantarum GBFG 61, which is deposited in the General Microbiology Center of the China Culture Collection Administration of Microorganisms with a deposit number of GGMCC NO: 31583 and a deposit date of August 14, 2024.

[0018] The Lactobacillus paracasei is Lactobacillus paracasei GBFG 34, which is deposited in the General Microbiology Center of the China Culture Collection Administration, with a deposit number of GGMCC NO: 31581 and a deposit date of August 14, 2024.

[0019] 3. The bacterial composition according to item 2, which can be used to ferment ginseng fermentation matrix to produce rare ginsenosides.

[0020] Here, the ginseng fermentation medium refers to a fermentation medium containing ginseng extract. The ginseng extract is obtained by reducing ginseng to smaller pieces, preferably using physical methods such as extrusion, cutting, or grinding. The ginseng extract can be, for example, ginseng powder. The ginseng fermentation medium may also contain other components commonly found in fermentation media, such as a nitrogen source, a carbon source, an inorganic salt, a pH adjuster, and the like.

[0021] 4. The bacterial composition according to item 2, wherein the dry weight ratio of Lactobacillus plantarum, Lactobacillus casei, Lactobacillus paracasei, and Bifidobacterium adolescens is: 1:(1-5):(1-5):(1-5).

[0022] 5. The bacterial composition according to item 2, wherein the Lactobacillus casei is Lactobacillus casei BL23.

[0023] 6. The bacterial composition according to item 2, wherein the Bifidobacterium adolescentis (Bifidobacterium adolescens) is Bifidobacterium adolescentis (Bifidobacterium adolescens) BH-J2356.

[0024] 7. A method for preparing a ginseng fermentation product, comprising inoculating a bacterial composition into a ginseng fermentation substrate and fermenting at 37-42°C for more than 30 hours to obtain a ginseng fermentation product containing rare ginsenosides;

[0025] Wherein, the bacterial composition is the bacterial composition described in any one of items 2 to 6.

[0026] 8. The preparation method according to item 7, further comprising sterilizing the ginseng fermentation product.

[0027] 9. The preparation method according to item 7, wherein the ginseng fermentation product is the ginseng fermentation product according to item 1.

[0028] 10. The preparation method according to item 7, wherein the ginseng fermentation product is fermented ginseng liquid or fermented ginseng powder.

[0029] 11. A ginseng fermentation product obtained by the preparation method of any one of items 7 to 10.

[0030] Figures in the specification

[0031] Figure 1 This is the high performance liquid chromatogram for the detection of fermented ginseng powder 2.

[0032] Figure 2 This is a high performance liquid chromatogram for detecting fermented ginseng powder 5. DETAILED DESCRIPTION

[0033] The present invention will be described in detail below with reference to specific embodiments. Unless otherwise specified, the scientific terms in this specification have the meanings commonly understood by those skilled in the art. In the event of a conflict, the definitions in this specification shall prevail.

[0034] Example

[0035] Example 1 Preparation of Fermented Ginseng Powder Rich in Rare Ginsenosides and Enhanced Anti-Fatigue Efficacy

[0036] The steps include:

[0037] (1) Pretreatment of ginseng fermentation matrix: 120 g ginseng powder, 10 g glucose, 2 g yeast extract powder, 2 g citric acid, 0.05 g manganese sulfate, and 0.1 g magnesium sulfate were dissolved in 856 g water and sterilized by autoclaving at 121°C for 20 min before use;

[0038] (2) Preparation of strains: Lactobacillus plantarum GBFG 61, Lactobacillus casei BL23, Lactobacillus paracasei GBFG 34, Bifidobacterium adolescentis BH-J2356, Lactobacillus plantarum Lp90, and Lactobacillus paracasei LC37 were inoculated into 5 mL of MRS broth culture medium and placed in a constant temperature incubator at 37°C ± 1°C for 24 h to obtain a first-level seed solution. Each bacterial solution was then inoculated into 50 mL of culture medium at a 1% inoculum volume and cultured for 24 h to obtain a second-level seed solution. The activity of each strain was 10 7 cfu / mL to 10 9 cfu / mL. The dry bacterial content in each of the above-mentioned second-level seed solutions was determined.

[0039] It should be noted that the above-mentioned Lactobacillus plantarum GBFG 61 is a lactobacillus strain that produces β-glucosidase and was isolated and cultivated from the soil of the ginseng planting base in Fusong County, Baishan City, Jilin Province. The lactobacillus strain was named GBFG 61 and was deposited in the General Microbiology Center of the China Culture Collection of Microorganisms with a deposit number of CGMCC No. 30101. The deposit date is March 22, 2024, and the deposit address is: Institute of Microbiology, Chinese Academy of Sciences, No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing; Postal code of the deposit institution is: 100101.

[0040] The above-mentioned Lactobacillus paracasei GBFG 34 is a lactobacillus strain that produces β-glucosidase and was isolated and cultivated from the soil of the ginseng planting base in Ji'an City (County) of Tonghua City, Jilin Province. The lactobacillus strain was named GBFG 34 and was deposited in the General Microbiology Center of the China Microorganism Culture Collection Administration Committee with a deposit number of CGMCC No.31581. The deposit date is August 14, 2024, and the deposit address is: Institute of Microbiology, Chinese Academy of Sciences, No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing; Postal code of the deposit institution is: 100101.

[0041] The above-mentioned Lactobacillus casei BL23 was purchased from Beijing Crespo Biotechnology Co., Ltd.; Bifidobacterium adolescens BH-J2356 was purchased from Shanghai Bohu Biotechnology Co., Ltd.; Lactobacillus plantarum Lp90 and Lactobacillus paracasei LC37 were purchased from Weikang Probiotics (Suzhou) Co., Ltd.

[0042] (3) Fermentation: The second-level seed liquid of Lactobacillus plantarum GBFG 61, Lactobacillus casei BL23, Lactobacillus paracasei GBFG 34, and Bifidobacterium adolescentis BH-J2356 were mixed to 10 g at a dry bacteria weight ratio of 1:1:1:1 (composition 1), 1:2:3:2 (composition 2), 1:3:2:3 (composition 3), and 1:5:5:5 (composition 4), and aseptically inoculated into ginseng fermentation matrix. The fermentation temperature was set to 37°C and the rotation speed was 50 rpm. The fermentation was carried out for 48 h, and the pH was maintained at 4-5 during the fermentation process.

[0043] For comparison, the Lactobacillus plantarum Lp90 was used to replace the Lactobacillus plantarum GBFG 61 in the composition 2 (composition 5); and the Lactobacillus paracasei LC37 was used to replace the Lactobacillus paracasei GBFG 34 in the composition 2 (composition 6).

[0044] (4) Centrifugation, drying, and crushing: After the fermentation is completed, sterilization, centrifugation, and drying operations are performed. The sterilization parameters are: 121°C, 20 min; the centrifugation parameters are: 3000-5000 rpm, 10 min; the vacuum degree is controlled to be ≤-0.09 MPa, 60-65°C, and the mixture is dried for 48 hours and then crushed to obtain fermented ginseng powders 1 to 6.

[0045] Detection and Analysis: Each of the above fermented ginseng powders was dissolved in methanol to prepare 80 mg / mL. 1 mL of each solution was taken and filtered through a 0.45 μm microporous filter membrane to obtain a clear solution. The total ginsenoside content and the content of various rare ginsenosides were determined, and the difference between the content of ginsenoside Rh4 (positive) related to anti-fatigue and the content of ginsenosides Rk1 (reverse) and Rg5 (reverse) was calculated (weight %, with the total ginsenoside amount as 100 weight %). The results are shown in Table 1.

[0046] The detection method of total ginsenosides was carried out according to the method of content determination under the item of total ginsenosides in the 2020 edition of the Pharmacopoeia of the People's Republic of China. The results were expressed as ginsenoside Re(C 48 H 82 O 18 )count.

[0047] The detection method of rare ginsenosides is carried out according to the method for determination of the content of rare ginsenosides in T / CNHFA 001-2021.

[0048] As an example, the HPLC analysis of the fermented ginseng powder 2 obtained by using the above composition 2 is as follows: Figure 1 As shown, the high performance liquid chromatography of the fermented ginseng powder 5 obtained by using the composition 5 is as shown in FIG. Figure 2 shown.

[0049] Table 1: Detection of rare ginsenoside content in fermented ginseng powder obtained using different bacterial strain compositions

[0050]

[0051] Difference = Ginsenoside Rh4 content - (Ginsenoside Rk1 content + Ginsenoside Rg5 content)

[0052] As shown in Table 1, the content of ginsenoside Rh4, which has an anti-fatigue effect, in fermented ginseng powders 1 to 4 obtained using Lactobacillus plantarum GBFG 61 and Lactobacillus paracasei GBFG 34 was 3% or more by weight, and the difference between the ginsenoside Rh4 content and (ginsenoside Rk1 content + ginsenoside Rg5 content) was -5% or more by weight. In contrast, the content of ginsenoside Rh4, which has an anti-fatigue effect, in fermented ginseng powders 5 and 6 obtained using other Lactobacillus plantarum and Lactobacillus paracasei strains was less than 3% by weight, and the difference between the ginsenoside Rh4 content and (ginsenoside Rk1 content + ginsenoside Rg5 content) was less than -5% by weight. It is expected that fermented ginseng powders 1 to 4 have better anti-fatigue effects than fermented ginseng powders 5 and 6.

[0053] It should also be noted that, in addition to the above-mentioned Lactobacillus plantarum Lp90 and Lactobacillus paracasei LC37 used for comparison, the inventors also tried a variety of Lactobacillus plantarum and Lactobacillus paracasei strains isolated by themselves, but none of them could achieve the technical effect of "the content of ginsenoside Rh4 is 3 weight% and the difference is greater than -0.5%".

[0054] Example 2 Verification of the Anti-fatigue Effect of Fermented Ginseng Powder

[0055] With reference to Example 6 of Chinese Patent CN 114762529 B, with slight modifications, the anti-fatigue effects of the fermented ginseng powder were tested, confirming that the anti-fatigue efficacy of fermented ginseng powder 2 was superior to that of fermented ginseng powder 5. It should be noted that while fermented ginseng powder 2 and fermented ginseng powder 5 had similar ginsenoside Rh4 contents, only fermented ginseng powder 2 met the requirement of a difference of at least -5% by weight.

[0056] Experimental Animals: 60 SPF-grade Kunming mice (30 males, 30 females), weighing 18-22 g, were randomly divided into three groups of 20 mice each (10 males, 10 females per group) and acclimated for 7 days. Experimental conditions: SPF animal room, temperature 20-26°C, humidity 40%-70%, 12-hour light / dark cycle. Dosage Grouping and Test Sample Administration Schedule: After the acclimation period, the three groups were divided into a blank control group, two fermented ginseng powder groups, and five fermented ginseng powder groups. Gavage administration of 250 mg / kg, 10 times the recommended human dose, was continued for 4 weeks. Experimental Methods: ① 30 minutes after the last administration of the test sample, the mice were placed in a swimming tank with a lead weight of 5% of their body weight at the base of their tails. The water depth was at least 20 cm and the temperature was 25°C ± 2°C. The time from the start of swimming to death (weighted swimming time) was measured for each group of 10 mice (10 per group). ② After the final administration, mice in each group swam weightlessly in 30°C water. Orbital blood was collected and serum urea nitrogen (BUN), blood lactate (LA), lactate dehydrogenase (LDH), and creatine phosphokinase (CPK) levels were measured in 10 mice per group. The livers were isolated and the liver glycogen (LG) content was measured. The results are shown in Table 2.

[0057] Table 2: Anti-fatigue animal test index detection of fermentation products of different bacterial species

[0058]

[0059] Note: Compared with the blank control group, ***P<0.001, **P<0.01, *P<0.05; compared with composition 3, ###P<0.001, ##P<0.01, #P<0.05

[0060] As shown in the table above, compared with the blank control group, the weighted swimming time of the mice in the fermented ginseng powder 2 group was significantly increased, indicating that the ginseng fermentation product of the present invention has anti-fatigue activity in prolonging the weighted swimming time of mice. The weighted swimming time of the mice in the fermented ginseng powder 2 group was significantly different from that in the fermented ginseng powder 5 group, indicating that the fermented ginseng powder 2 has a stronger anti-fatigue activity in prolonging the weighted swimming time of mice than the fermented ginseng powder 5 group.

[0061] In terms of serum and liver test indicators, the blood lactate (LA), urea nitrogen (BUN), and lactate dehydrogenase (LDH) levels of mice in the blank control group, fermented ginseng powder 2, and fermented ginseng powder 5 groups were significantly reduced, and the liver glycogen (LG) content and creatine phosphokinase (CPK) were significantly increased, indicating that both fermented ginseng powder samples have anti-fatigue effects. This proves that the fermented ginseng powder of the embodiment of the present invention may exert its anti-fatigue activity by reducing the content of LA and BUN, reducing LDH activity, and increasing the content of LG and CPK. Moreover, compared with fermented ginseng powder 5, fermented ginseng powder 2 has significant differences in BUN, LDH, and CPK content.

[0062] Based on the above test results, it can be seen that compared with the comparative fermented ginseng powder 5 (difference less than -0.5 weight %), the fermented ginseng powder 2 of the present invention (difference greater than -0.5 weight %) has significantly stronger anti-fatigue effect.

[0063] It should be noted that, provided that it is feasible and does not significantly violate the main purpose of the present invention, any technical feature or combination of technical features described in this specification as a component of a technical solution may also be applied to other technical solutions; and, provided that it is feasible and does not significantly violate the main purpose of the present invention, technical features described as components of different technical solutions may also be combined in any manner to form other technical solutions. The present invention also includes technical solutions obtained through the above-mentioned combinations, and these technical solutions are equivalent to those described in this specification.

[0064] The present invention has been described above through specific implementation methods and examples, but those skilled in the art should understand that these are not intended to limit the scope of the present invention, and the scope of the present invention should be determined by the claims.

[0065] Industrial Applicability

[0066] The present invention provides a ginseng fermentation product containing rare ginsenosides and having enhanced anti-fatigue effect, as well as a preparation method and application thereof.

Claims

1. A bacterial composition, which can be used to ferment ginseng fermentation matrix to produce rare ginsenosides, which is composed of Lactobacillus plantarum, Lactobacillus casei, Lactobacillus paracasei and Bifidobacterium adolescentis; wherein, The plant lactobacillus is Lactobacillus plantarum GBFG 61, which is deposited in the General Microbiology Center of the China Culture Collection Administration of Microorganisms with a deposit number of GGMCCNO: 31583 and a deposit date of August 14, 2024. The Lactobacillus paracasei is Lactobacillus paracasei GBFG 34, which is deposited in the General Microbiology Center of the China Culture Collection Administration of Microorganisms with a deposit number of GGMCC NO: 31581 and a deposit date of August 14, 2024. The Lactobacillus casei is Lactobacillus casei BL23; and The adolescent bifidobacterium is Bifidobacterium adolescens BH-J2356; The dry weight ratio of Lactobacillus plantarum, Lactobacillus casei, Lactobacillus paracasei and Bifidobacterium adolescence is 1:1:1:1 or 1:2:3:2 or 1:3:2:3 or 1:5:5:

5.

2. A method for preparing a ginseng fermentation product, comprising inoculating a strain composition into a ginseng fermentation substrate and fermenting at 37°C for 48 hours at a rotation speed of 50 rpm, maintaining a pH of 4-5 during the fermentation process, thereby obtaining a ginseng fermentation product containing rare ginsenosides; in, The bacterial composition is the bacterial composition according to claim 1.

3. The preparation method according to claim 2, wherein The ginseng fermentation product comprises ginsenoside Rh4, ginsenoside Rk1 and ginsenoside Rg5, which meet the following requirements: The content of ginsenoside Rh4 is ≥3% by weight; and Ginsenoside Rh4 content - Ginsenoside Rk1 content - Ginsenoside Rg5 content ≥ - 5% by weight The total amount of saponins in the ginseng fermentation product is taken as 100% by weight.

4. The preparation method according to claim 2, wherein The ginseng fermentation product is fermented ginseng liquid or fermented ginseng powder.

5. A ginseng fermentation product prepared by the preparation method according to any one of claims 2 to 4, comprising ginsenoside Rh4, ginsenoside Rk1 and ginsenoside Rg5, which satisfies: The content of ginsenoside Rh4 is ≥3% by weight; and Ginsenoside Rh4 content - Ginsenoside Rk1 content - Ginsenoside Rg5 content ≥ - 5% by weight The total amount of saponins in the ginseng fermentation product is taken as 100% by weight.

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