Ginseng fermentation product rich in ginsenosides Rg3, Rk1 and Rg5

Abstract

This patent application discloses a ginseng fermentation product rich in ginsenosides Rg3, Rk1 and Rg5, 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, enhancing the body's immunity, etc., 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 Rg3, Rk1 and Rg5, and the relative content of ginsenoside Rg3 is high. 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 effect of alleviating cognitive impairment. 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 belongs to the technical field of synthetic biology, and specifically relates to a ginseng fermentation product with enhanced efficacy in alleviating cognitive impairment and rich in rare ginsenosides Rg3, Rk1 and Rg5, as well as a preparation method and application thereof. Background Art

[0002] Ginseng is a traditional Chinese herbal medicine. Ginsenosides are widely recognized as active ingredients due to their well-defined medicinal properties. Ginsenosides Ra, Rb, Rc, Rd, Re, Rf, and Rg1 are inherent in ginseng and are present in high concentrations, accounting for over 95% of the total saponins. Rare ginsenosides are not naturally present in the plant and are only found in processed ginseng varieties, such as red and black ginseng. Due to their extremely low concentrations, they are therefore referred to as rare ginsenosides.

[0003] Ginsenosides have structural characteristics such as a large relative molecular mass, a small lipid-water partition coefficient, and a large topological polar surface area. Their bioavailability is low, and after oral administration, they must be metabolized by the intestinal flora in the gastrointestinal tract into secondary saponins, such as rare ginsenosides, before they can be absorbed and become active substances that truly exert their pharmacological effects. Currently, more than 60 rare ginsenosides have been discovered, including Rk2, Rg3, Rh2, Rg5, Rh1, Rh3, Rh4, and Rk1, which have high pharmacological activity in anti-tumor, anti-inflammatory, and immune-enhancing activities.

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

[0005] However, a technical problem with these existing technologies is that microbial fermentation produces a variety of rare ginsenoside compositions, which can include dozens of rare ginsenosides. These rare ginsenosides have varying biological effects, and some even antagonize each other. Consequently, these ginseng fermentation products are more like a "jack of all trades," potentially offering benefits in areas like skin whitening and immune enhancement, but without a single, overtly specific benefit.

[0006] In terms of the physiological effects of rare ginsenosides, the three ginsenosides Rg3, Rk1, and Rg5 are all believed to have certain effects in alleviating cognitive impairment. Ginsenosides Rk1 and Rg5 also have the function of inhibiting nerve excitement and helping sleep. Large amounts of intake may cause drowsiness in the subjects, especially the elderly and children, who are the groups that have a greater need to alleviate cognitive impairment. Summary of the Invention

[0007] In view of the technical problems existing in the above-mentioned prior art, the object of the present invention is to provide a ginseng fermentation product rich in rare ginsenosides, which has enhanced efficacy in alleviating cognitive impairment and is less likely to cause drowsiness, as well as a preparation method and application thereof.

[0008] After literature research, the inventors discovered that among the rare ginsenosides, ginsenosides Rg3, Rk1, and Rg5 are most effective in alleviating cognitive impairment. Furthermore, ginsenosides Rk1 and Rg5 can inhibit neural excitation and promote sleep. Based on this, they believe that by increasing the combined content of ginsenosides Rg3, Rk1, and Rg5 in a ginseng fermentation product, and simultaneously increasing the relative content of ginsenoside Rg3 among these three ginsenosides, it would be possible to produce a ginseng fermentation product with enhanced cognitive impairment-alleviating efficacy and reduced drowsiness.

[0009] 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 the three rare ginsenosides Rg3, Rk1, and Rg5, ginsenoside Rg5 is easier to produce, and often when a certain amount of ginsenosides Rg3 and Rk1 are produced, more ginsenoside Rg5 will be produced. This makes the existing ginseng fermentation products either have a low total content of the three ginsenosides Rg3, Rk1, and Rg5, or a low relative content of ginsenoside Rg3, making it difficult for the ginseng fermentation products to alleviate cognitive impairment while not easily causing drowsiness.

[0010] Furthermore, after conducting in-depth research to address the aforementioned technical issues, the inventors discovered that by enzymatically hydrolyzing the ginseng extract with a complex enzyme system consisting of cellulase, pectinase, acid protease, and β-glucosidase, followed by fermentation using a composite strain of a specific strain of Bifidobacterium animalis and Lactobacillus mucilaginosus, the inherent saponins in ginseng can be efficiently converted into the three rare saponins, while also increasing the relative content of ginsenoside Rg3. This results in a ginseng fermentation product that has enhanced cognitive impairment relief and is less prone to drowsiness.

[0011] The technical solution of the present invention includes:

[0012] 1. A ginseng fermentation product obtained by fermenting a ginseng fermentation substrate using a microbial strain, comprising three ginsenosides, namely, Rg3, Rk1, and Rg5, and satisfying the following requirements:

[0013] The total content of the ginsenosides Rg3, Rk1 and Rg5 is ≥35% by weight, with the total weight of the ginsenosides in the ginseng fermentation product being 100% by weight; and

[0014] Among the ginsenosides Rg3, Rk1 and Rg5, ginsenoside Rg3 is ≥40% by weight, and the total weight of the ginsenosides Rg3, Rk1 and Rg5 is 100% by weight.

[0015] 2. A bacterial composition consisting of Bifidobacterium animalis and Lactobacillus fermentans; wherein the Bifidobacterium animalis is Bifidobacterium animalis GBFG 52, which is deposited in the General Microbiology Center of the China Culture Collection Administration of Microorganisms, with a deposit number of CGMCC No.31582 and a deposit date of August 14, 2024.

[0016] 3. The bacterial composition according to item 2, which can be used to ferment ginseng fermentation matrix to produce rare ginsenosides.

[0017] 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.

[0018] 4. The bacterial composition according to item 2, wherein the dry weight ratio of Bifidobacterium animalis to Lactobacillus mucinous fermentation is 0.4 to 2:1.

[0019] 5. The bacterial composition according to item 2, wherein the fermentative Lactobacillus muciniphila is fermentative Lactobacillus muciniphila PG-LFF246.

[0020] 6. A method for preparing a ginseng fermentation product, comprising inoculating a bacterial composition into a ginseng enzymatic hydrolyzate and fermenting the mixture at 35-45°C, preferably 35-40°C, for 60-80 hours, preferably 65-75 hours, to obtain a ginseng fermentation product containing rare ginsenosides;

[0021] Wherein, the bacterial composition is the bacterial composition described in any one of items 2 to 5.

[0022] 7. The preparation method according to item 6, further comprising sterilizing the ginseng fermentation product.

[0023] 8. The preparation method according to item 6, wherein the ginseng fermentation product is the ginseng fermentation product according to item 1.

[0024] 9. The preparation method according to item 6, wherein the ginseng fermentation product is ginseng fermentation liquid or ginseng fermentation powder.

[0025] 10. The preparation method according to item 6, wherein the ginseng enzymatic hydrolyzate is prepared by enzymatically hydrolyzing the fermentation homogenate using an enzymatic agent;

[0026] The enzymatic agent comprises cellulase, pectinase, acid protease and β-glucosidase;

[0027] The fermentation slurry comprises ginseng powder and water.

[0028] 11. The preparation method according to item 10, wherein the weight ratio of the cellulase, pectinase, acid protease and β-glucosidase in the enzymatic agent is 1:1:1:1.

[0029] 12. The preparation method according to item 10, wherein

[0030] Ginseng powder is added with water to prepare a fermentation homogenate, and citric acid is added to adjust the pH of the fermentation homogenate to 3-5;

[0031] The enzymatic hydrolyzing agent is added to the fermentation homogenate, and enzymatic hydrolysis is carried out at 53-63° C. for 150-180 minutes, and the pH is adjusted to 4-5 to obtain the ginseng enzymatic hydrolyzate.

[0032] 13. The preparation method according to item 6, wherein

[0033] The ginseng enzymatic hydrolysate is kept at 80-90° C. for 40-60 minutes to obtain a sterilized ginseng enzymatic hydrolysate;

[0034] The sterilized ginseng hydrolysate is cooled to 35-45° C., preferably 35-40° C., and the bacterial composition is added thereto. The hydrolysate is fermented at 35-45° C., preferably 35-40° C., for 60-80 hours, preferably 65-75 hours to obtain a ginseng fermentation product.

[0035] 14. A ginseng fermentation product prepared by the method of any one of items 6 to 13, comprising three ginsenosides: Rg3, Rk1, and Rg5;

[0036] The total content of the three ginsenosides is 35% by weight or more, with the total weight of the ginsenosides in the ginseng fermentation product being 100% by weight; and

[0037] Among the ginsenosides Rg3, Rk1 and Rg5, ginsenoside Rg3 is ≥40% by weight, and the total weight of the ginsenosides Rg3, Rk1 and Rg5 is 100% by weight. BRIEF DESCRIPTION OF THE DRAWINGS

[0038] Figure 1 This is a high performance liquid chromatogram showing the determination of the contents of three ginsenosides in the ginseng fermentation broth prepared in Example 2.

[0039] Figure 2 This is a high performance liquid chromatogram of the determination of the contents of three ginsenosides in the ginseng fermentation broth prepared in Comparative Example 1. DETAILED DESCRIPTION

[0040] 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.

[0041] Example

[0042] The Bifidobacterium animalis is a β-glucosidase-producing Bifidobacterium strain isolated and cultivated from the soil of a ginseng planting base in Changbai County, Changbai Korean Autonomous County, Baishan City, Jilin Province, and is named Bifidobacterium animalis GBFG 52. It is deposited in the General Microbiology Center of the China Culture Collection of Microorganisms, with a deposit number of CGMCC No. 31582; the deposit date is August 14, 2024; the deposit address is: Institute of Microbiology, Chinese Academy of Sciences, No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing; the deposit postal code is: 100101.

[0043] The fermented mucus lactobacillus (Limosilactobacillus fermentum) PG-LFF246 was purchased from Zhejiang Yicun Biotechnology Co., Ltd.

[0044] Example 1 The preparation of a ginseng fermented liquid rich in rare ginsenosides and having enhanced efficacy in alleviating cognitive impairment comprises the following steps:

[0045] Extract ginseng powder with water at 80-85°C for 2 hours (the weight ratio of ginseng to water is 1:10), prepare a fermentation slurry, add citric acid, and adjust the pH to 4-5;

[0046] An enzymatic hydrolyzing agent (cellulase, pectinase, acid protease and β-glucosidase in a weight ratio of 1:1:1:1) is added to the fermentation homogenate, and enzymatic hydrolysis is carried out at 53-63° C. for 160 minutes, and the pH is adjusted to 4-5 to obtain an enzymatic hydrolyzate; the weight ratio of the enzymatic hydrolyzing agent to the ginseng powder is 1:10.

[0047] The enzymatic hydrolyzate is kept at 80-90° C. for 60 min to obtain a sterilized solution;

[0048] The sterilized liquid was cooled to 35-40° C., and composite fermentation bacterial powder (the dry weight ratio of Bifidobacterium animalis GBFG 52 and Lactobacillus mucinous fermentation PG-LFF246 was 0.4:1) was added to the sterilized liquid with an inoculation amount of 0.4% by weight. The mixture was fermented at 35-40° C. for 72 hours to obtain a fermentation liquid, which was freeze-dried to obtain Composition 1.

[0049] Example 2

[0050] The fermentation preparation was carried out as in Example 1 to obtain Composition 2, except that the dry weight ratio of Bifidobacterium animalis GBFG 52 to Lactobacillus mucilaginosus PG-LFF246 in the composite fermentation bacterial powder was 1:1.

[0051] Example 3

[0052] The fermentation preparation was carried out in the same manner as in Example 1 to obtain Composition 3, except that the dry weight ratio of Bifidobacterium animalis GBFG 52 to Lactobacillus mucilaginosus PG-LFF246 in the composite fermentation bacterial powder was 2:1.

[0053] Comparative Example 1

[0054] The fermentation preparation was carried out as in Example 2 to obtain a comparative composition 1, except that the Bifidobacterium animalis GBFG 52 was replaced by Bifidobacterium animalis BB-12 (purchased from Shaanxi Angxu Biotechnology Co., Ltd.).

[0055] Comparative Example 2

[0056] The fermentation preparation was carried out as in Example 2 to obtain a comparative composition 2, except that the fermented Lactobacillus muciniphila PG-LFF246 was replaced by fermented Lactobacillus muciniphila CECT5716 (purchased from Shaanxi Angxu Biotechnology Co., Ltd.).

[0057] Comparative Example 3

[0058] The fermentation preparation was carried out in the same manner as in Example 2 to obtain Comparative Composition 3, except that the fermentation homogenate was directly sterilized before fermentation without the enzymatic hydrolysis step.

[0059] Example 4 Determination of rare ginsenoside content in each composition

[0060] Each of the above compositions was taken separately, freeze-dried, and then dissolved in methanol to prepare 80 mg / mL. 1 mL of the solution was taken separately and filtered with a 0.45 μm microporous filter membrane to obtain a clear solution. The total ginsenoside content and the content of various rare ginsenosides therein were determined, and the total amount of the three rare ginsenosides Rg3, Rk1 and Rg5 (weight %, based on the total ginsenoside amount as 100 weight %) and the relative content of ginsenoside Rg3 in the three rare ginsenosides Rg3, Rk1 and Rg5 (based on the total amount of the three rare ginsenosides Rg3, Rk1 and Rg5 as 100 weight %) were calculated.

[0061] The results are shown in Table 1.

[0062] The detection method of total saponins 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.

[0063] The detection method for rare ginsenosides is carried out according to the method for determination of the content of rare ginsenosides in T / CNHFA001-2021.

[0064] As an example, the HPLC chromatograms for determining the total content of the three ginsenosides Rg3, Rk1 and Rg5 in the compositions prepared in Example 2 and Comparative Example 1 are shown as follows: Figure 1 and Figure 2 shown.

[0065] Table 1 Detection results of rare ginsenoside contents in different ginsenoside compositions

[0066]

[0067] As shown in Table 1, the total content of the three rare ginsenosides Rg3, Rk1, and Rg5 in Compositions 1 to 3 reached 35% by weight or more, and the relative content of ginsenoside Rg3 reached 40% by weight or more. This suggests that the compositions have better efficacy in alleviating cognitive impairment.

[0068] Example 5 Effects of the Compositions on the Scopolamine-Induced Memory Impairment in Mice in the Platform-Stepping Test

[0069] Fifty mice were randomly divided into a control group, a model group, a piracetam group, a combination group 2, and a comparative combination group 1. After 30 days of gavage (specific dosages are shown in the table below), a formal experiment was conducted. 10 minutes before training, except for the control group, which received an equal amount of normal saline intraperitoneally, all other groups received an intraperitoneal injection of scopolamine 5 mg / kg. The number of times the experimental animals jumped off the platform within 5 minutes of the test period was observed and recorded as the "number of errors" and the latency period of the test period. The differences in memory performance of mice in different groups were compared.

[0070] The platform jumping test observed a significant increase in the number of incorrect responses in the scopolamine model group compared to the control group, and a significant decrease in the latency to electric shock during the test period, indicating successful model establishment. Mice in the Composition 2 group showed a significant decrease in incorrect platform jumping responses during the test period (P < 0.01) and a significant increase in the latency to electric shock during the test period (P < 0.01) compared to the model group (Table 2).

[0071] Table 2 Effects of ginsenoside composition on learning and memory ability of mice in platform jumping test ( n=10)

[0072]

[0073] Note: Compared with the control group, #: P < 0.05, ##: P < 0.01; compared with the model group, *: P < 0.05, **: P < 0.01 Example 6 Effects of each composition on the water maze test of mice with memory impairment caused by scopolamine

[0074] Fifty mice were randomly divided into a control group, a model group, a piracetam group, a composition group 2, and a comparative composition group 1. After 30 days of gavage (specific dosages are shown in the table below), a formal experiment was conducted. 10 minutes before training, except for the control group, which was intraperitoneally injected with an equal amount of normal saline, all other groups were intraperitoneally injected with scopolamine 5 mg / kg. Samples were continued during training, once a day. The water depth of the maze lane was 9 cm, and the water temperature was about 20°C. The training time for mice was limited to 2 minutes, and mice that did not reach the end point within 2 minutes were counted as 2 minutes. Finally, the total number of errors in 5 training and tests for each group of animals, the total time to reach the end point, and the total number of animals that reached the end point within 2 minutes were calculated.

[0075] After scopolamine administration, the escape latency of mice in the model group was significantly prolonged, with a highly significant difference between the model group and the control group (P < 0.01), indicating successful model establishment and a stable and reliable scopolamine-induced memory impairment model in mice. Compared with the model group, mice administered the ginsenoside composition of Composition 2 showed varying degrees of reduction in escape latency and number of platform crossings (P < 0.05 or P < 0.01). This trend became increasingly pronounced over time (see Table 3).

[0076] Table 3 Comparison of latency and number of crossing platforms in water maze test among mice in each group ( n=10)

[0077]

[0078] Note: Compared with the control group, #: P < 0.05, ##: P < 0.01; compared with the model group, *: P < 0.05, **: P < 0.01

[0079] As shown in Tables 2 and 3, the ginseng fermentation product of the present invention has a high total content of the three ginsenosides Rg3, Rk1 and Rg5, and has a significant improvement effect on the memory function of memory impairment model mice, which suggests that it has the potential to treat cognitive decline.

[0080] 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.

[0081] Industrial Applicability

[0082] The present invention provides a ginseng fermentation product containing rare ginsenosides and having enhanced efficacy in alleviating cognitive impairment, as well as a preparation method and application thereof.

Claims

1. A bacterial composition for fermenting ginseng fermentation substrate to produce rare ginsenosides, the bacterial composition consisting of Bifidobacterium animalis and Lactobacillus mucilaginosus fermentation; wherein, The Bifidobacterium animalis is Bifidobacterium animalis GBFG 52, which is deposited in the General Microbiology Center of China Culture Collection Administration of Microorganisms, with a deposit number of CGMCC No. 31582 and a deposit date of August 14, 2024; The fermented mucus lactobacillus is fermented mucus lactobacillus (Limosilactobacillus fermentum) PG-LFF246.

2. The use according to claim 1, wherein The dry weight ratio of the animal bifidobacterium to the fermented mucus lactobacillus is 0.4 to 2:

1.

3. A method for preparing a ginseng fermentation product, comprising inoculating a bacterial composition into a ginseng enzymatic hydrolyzate and fermenting the mixture at 35-45°C for 60-80 hours to obtain a ginseng fermentation product containing rare ginsenosides; in, The bacterial composition is used to ferment ginseng fermentation matrix to produce rare ginsenosides, and is composed of Bifidobacterium animalis and Lactobacillus fermentum; wherein the Bifidobacterium animalis is Bifidobacterium animalis GBFG 52, which is deposited in the General Microbiology Center of the China Microorganism Culture Collection Administration with a deposit number of CGMCC No. 31582 and a deposit date of August 14, 2024; and the Lactobacillus fermentum is Lactobacillus fermentum PG-LFF246; The ginseng fermentation product contains three ginsenosides, namely Rg3, Rk1 and Rg5; The total content of the three ginsenosides is 35% by weight or more, with the total weight of the ginsenosides in the ginseng fermentation product being 100% by weight; and Among the ginsenosides Rg3, Rk1 and Rg5, ginsenoside Rg3 is ≥40% by weight, and the total weight of the ginsenosides Rg3, Rk1 and Rg5 is 100% by weight.

4. The preparation method according to claim 3, wherein The ginseng fermentation product is ginseng fermentation liquid or ginseng fermentation powder.

5. The preparation method according to claim 3, wherein The ginseng enzymatic hydrolysate is prepared by enzymatically hydrolyzing the fermentation homogenate using an enzymatic agent; The enzymatic agent comprises cellulase, pectinase, acid protease and β-glucosidase; The fermentation slurry comprises ginseng powder and water.

6. The preparation method according to claim 5, wherein In the enzymatic agent, the weight ratio of the cellulase, pectinase, acid protease and β-glucosidase is 1:1:1:

1.

7. The preparation method according to claim 5, wherein Ginseng powder is added with water to prepare a fermentation homogenate, and citric acid is added to adjust the pH of the fermentation homogenate to 3-5; The enzymatic hydrolyzing agent is added to the fermentation homogenate, and enzymatic hydrolysis is carried out at 53-63° C. for 150-180 minutes, and the pH is adjusted to 4-5 to obtain the ginseng enzymatic hydrolyzate.

8. The preparation method according to claim 3, wherein keeping the ginseng hydrolysate at 80-90° C. for 40-60 minutes to obtain a sterilized ginseng hydrolysate; and The sterilized ginseng enzymatic hydrolysate is cooled to 35-45° C., the bacterial composition is added thereto, and fermented at 35-45° C. for 60-80 hours to obtain a ginseng fermentation product.

9. The preparation method according to claim 8, wherein The sterilized ginseng enzymatic hydrolysate is cooled to 35-40° C., the bacterial composition is added thereto, and fermented at 35-40° C. for 65-75 hours to obtain a ginseng fermentation product.

10. The preparation method according to claim 3, wherein The bacterial composition is inoculated into ginseng enzymatic hydrolysate and fermented at 35-40° C. for 65-75 hours to obtain a ginseng fermentation product containing rare ginsenosides.

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