Ginseng fermentation product rich in ginsenosides Rg3, Rk1 and Rg5

Abstract

The invention discloses a ginseng fermentation product rich in ginsenosides Rg3, Rk1 and Rg5 as well as a preparation method and application of the ginseng fermentation product. Aiming at the problem that the existing ginseng fermentation product has certain effects in the aspects of whitening, resisting anemia, resisting inflammation, delaying cognitive impairment, enhancing the immunity of the organism and the like, but no effect is particularly clear and prominent, the ginseng fermentation product is prepared by fermenting a ginseng fermentation substrate by using a specific probiotic composition; the composition is rich in rare ginsenosides Rg3, Rk1 and Rg5, and the relative content of the ginsenosides Rg3 is high. Experimental research shows that compared with the existing ginseng fermentation product, the ginseng fermentation product disclosed by the invention has a more definite and prominent effect of relieving cognitive impairment. The ginseng fermentation product is simple in preparation method and easy to industrialize, can be widely applied to functional food and other large health fields, and has a wide application prospect.

Description

Technical Field

[0001] The present invention belongs to the technical field of synthetic biology, and specifically relates to a ginseng fermentation product having enhanced efficacy in alleviating cognitive impairment and rich in rare ginsenosides Rg3, Rk1 and Rg5, and a preparation method and application thereof. Background Art

[0002] Ginseng is a traditional Chinese herbal medicine. Ginsenosides are widely accepted as effective ingredients for their clear functional efficacy. Ginsenosides Ra, Rb, Rc, Rd, Re, Rf and Rg1 are inherent saponins in ginseng, and their content is relatively high in ginseng, accounting for more than 95% of the total saponins. Rare ginsenosides are not natural components of plants, but only exist in processed ginseng such as red ginseng and black ginseng. Because of their extremely low content, they are called rare ginsenosides.

[0003] Ginsenosides have structural characteristics such as large relative molecular mass, small lipid-water partition coefficient, and large topological polar surface area. They have low bioavailability. After oral administration, they need to be metabolized by intestinal flora in the gastrointestinal tract into secondary saponin components such as rare ginsenosides before they can be absorbed and become active substances that truly exert their medicinal effects. At present, more than 60 rare ginsenosides have been discovered, including Rk2, Rg3, Rh2, Rg5, Rh1, Rh3, Rh4, Rk1, etc., which have high pharmacological activities in anti-tumor, anti-inflammatory, and immunity enhancement.

[0004] In the research on the production technology of rare ginsenosides, the biotransformation method of ginseng fermented by microorganisms is considered to be one of the safest and most efficient transformation methods for ginsenosides in vitro. In fact, technicians have used one microorganism or a combination of multiple microorganisms to ferment ginseng fermentation matrix to produce a variety of rare ginsenosides.

[0005] However, the technical problem of these existing technologies is that the microbial fermentation produces a combination of multiple rare ginsenosides, which may contain up to dozens of rare ginsenosides, and the biological effects of these rare ginsenosides are different, and the biological effects of some rare ginsenosides are even antagonistic to each other. Therefore, such ginseng fermentation products are more like "panacea", which may have some effects in aspects such as whitening and enhancing the body's immunity, but there is no particularly clear and prominent effect in any aspect.

[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, while ginsenosides Rk1 and Rg5 also have the function of inhibiting nerve excitement and aiding sleep. Large intake may cause drowsiness in the subjects, especially the elderly and children, who are the groups with 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 effect in alleviating cognitive impairment and is less likely to cause drowsiness, and a preparation method and application thereof.

[0008] The inventors have found through literature research that among the rare ginsenosides, ginsenosides Rg3, Rk1, and Rg5 can alleviate cognitive impairment, and ginsenosides Rk1 and Rg5 can also inhibit nerve excitation and promote sleep. Based on this, it can be considered that if the total content of ginsenosides Rg3, Rk1, and Rg5 in ginseng fermentation products can be increased, and the relative content of ginsenoside Rg3 in these three ginsenosides can be increased, a ginseng fermentation product with enhanced cognitive impairment alleviation effect and less prone to drowsiness can be prepared.

[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 path. 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, resulting in the ginseng fermentation products being difficult to alleviate cognitive impairment while not easily causing drowsiness.

[0010] Furthermore, the inventors conducted in-depth research to solve the above technical problems and found that by first enzymolyzing the ginseng extract with a composite enzyme of cellulase, pectinase, acid protease and β-glucosidase, and then fermenting it with a composite strain of specific animal bifidobacterium and fermented mucus lactobacillus, the inherent saponins in ginseng can be efficiently converted into the above three rare saponins, and the relative content of ginsenoside Rg3 can be increased. Thus, a ginseng fermentation product with enhanced efficacy in relieving cognitive impairment and less prone to drowsiness is prepared.

[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:

[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, which is composed of animal Bifidobacterium and fermented mucus Lactobacillus; wherein the animal Bifidobacterium (Bifidobacterium animalis) is animal Bifidobacterium GBFG 52, which is deposited in the General Microbiological Center of the China Microbiological Culture Collection Administration, 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 substrate to produce rare ginsenosides.

[0017] Here, the ginseng fermentation matrix refers to a fermentation matrix containing ginseng fines. The ginseng fines are obtained by reducing ginseng, preferably by physical methods such as extrusion, cutting, grinding, etc. The ginseng fines can be, for example, ginseng powder. The ginseng fermentation matrix can also contain other components commonly contained in the fermentation matrix, such as nitrogen source, carbon source, inorganic salt, pH regulator, etc.

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

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

[0020] 6. A method for preparing a ginseng fermentation product, comprising inoculating a bacterial composition into a ginseng hydrolysate, and fermenting 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 enzymolysis agent comprises cellulase, pectinase, acid protease and β-glucosidase;

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

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

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

[0030] Adding water to ginseng powder to prepare a fermentation slurry, adding citric acid, and adjusting the pH of the fermentation slurry to 3-5;

[0031] The enzymolysis agent is added to the fermentation homogenate, and enzymolysis is performed at 53-63° C. for 150-180 minutes, and the pH is adjusted to 4-5 to obtain the ginseng enzymolysis solution.

[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, and 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 preparation method described in any one of items 6 to 13, comprising three ginsenosides, namely, 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 The HPLC chromatogram is for determining 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 fermented liquid prepared in Comparative Example 1. DETAILED DESCRIPTION

[0040] The present invention is described in detail below in conjunction with specific embodiments. In the absence of conflict, the scientific terms in this specification have the meanings commonly understood by those skilled in the art. If there is 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 the 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 Microbiological Center of the China Microbiological Culture Collection Administration, and the deposit number is CGMCC No.31582; ​​the deposit date is August 14, 2024; the deposit institution address is: No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing, Institute of Microbiology, Chinese Academy of Sciences; the deposit institution 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 cognitive impairment relieving efficacy 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 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 fermented bacterial powder (the dry weight ratio of animal Bifidobacterium GBFG 52 to fermented Lactobacillus mucosa PG-LFF246 was 0.4:1) was added to the sterilized liquid, with an inoculation amount of 0.4% by weight, and fermented at 35-40° C. for 72 hours to obtain a fermented liquid, which was freeze-dried to obtain Composition 1.

[0049] Example 2

[0050] The fermentation preparation was carried out in the same manner 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 fermented 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 fermented 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 animal Bifidobacterium GBFG 52 was replaced by animal Bifidobacterium 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 mucoides PG-LFF246 was replaced by fermented Lactobacillus mucoides 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 and then fermented 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, and the total ginsenoside content and the content of various rare ginsenosides therein were determined. The total amount of the three rare ginsenosides Rg3, Rk1 and Rg5 (weight %, taking the total ginsenoside amount as 100 weight %) and the relative content of ginsenoside Rg3 in the three rare ginsenosides Rg3, Rk1 and Rg5 (taking 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 for determining the content of total ginsenosides in the Pharmacopoeia of the People's Republic of China (2020 edition). 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 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 the compositions 1 to 3 is more than 35 wt%, and the relative content of ginsenoside Rg3 is more than 40 wt%, which indicates that the composition has a better effect in alleviating cognitive impairment.

[0068] Example 5 Effects of various compositions on the platform jumping test of mice with memory impairment induced by scopolamine

[0069] 50 mice were randomly divided into control group, model group, piracetam group, combination group 2, and comparison combination group 1. After 30 days of intragastric administration (specific doses are shown in the following table), formal experiments were conducted. Except for the control group, which was intraperitoneally injected with an equal amount of saline 10 minutes before training, all other groups were intraperitoneally injected with 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 of the test period, and the differences in memory performance of mice between different groups were compared.

[0070] The jumping platform experiment observed that the number of wrong responses in the scopolamine model group was significantly increased compared with the normal control group, and the electric shock latency during the test period was significantly shortened, indicating that the model was successfully established. Compared with the model group, the number of wrong responses in the jumping platform during the test period in the combination group 2 was significantly reduced (P < 0.01) and the electric shock latency during the test period was significantly prolonged (P < 0.01) (see Table 2).

[0071] Table 2 Effects of ginsenoside composition on learning and memory ability of mice in stepping platform 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 combination group 2, and a comparative combination group 1. After 30 days of intragastric administration (the specific dosage is as shown in the table below), a formal experiment was conducted. Except for the control group, which was intraperitoneally injected with an equal amount of normal saline 10 minutes before training, all other groups were intraperitoneally injected with scopolamine 5 mg / kg. Samples continued to be given 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 recorded 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 was administered, the escape latency of mice in the model group was significantly prolonged, and the difference between the model group and the control group was very significant (P < 0.01), indicating that the model was successfully established and the scopolamine-induced mouse memory impairment model was stable and reliable. Compared with the model group, the escape latency and the number of platform crossings of mice administered with the ginsenoside composition of composition 2 were reduced to varying degrees (P < 0.05 or P < 0.01). Moreover, the trend became more and more obvious as time went on (see Table 3).

[0076] Table 3 Comparison of latency and number of crossing platforms in water maze test of 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 combined content of the three ginsenosides Rg3, Rk1 and Rg5 in the ginseng fermentation product of the present invention is high, and has a significant improvement effect on the memory function of memory impairment model mice, which indicates that it has the potential to treat cognitive decline.

[0080] The present invention is described above through specific implementation modes 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 effect of alleviating cognitive impairment, and a preparation method and application thereof.

Claims

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 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 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.

2. A bacterial composition, which consists of animal Bifidobacterium and fermented Lactobacillus mucus; wherein, The Bifidobacterium animalis is Bifidobacterium animalis GBFG 52, which is deposited in the General Microbiological Center of China Microbiological Culture Collection Administration, 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.

3. The bacterial composition according to claim 2, wherein The dry weight ratio of the animal bifidobacterium to the fermented mucus lactobacillus is 0.4-2:

1.

4. A method for preparing a ginseng fermentation product, comprising inoculating a bacterial composition into a ginseng hydrolysate, and fermenting 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; in, The bacterial composition is the bacterial composition according to any one of claims 2 or 3.

5. The preparation method according to item 4, wherein: The ginseng fermentation product is ginseng fermentation liquid or ginseng fermentation powder.

6. The preparation method according to item 4, wherein: The ginseng enzymatic hydrolysate is prepared by enzymatically hydrolyzing the fermentation homogenate using an enzymatic agent; The enzymolysis agent comprises cellulase, pectinase, acid protease and β-glucosidase; The fermented slurry comprises ginseng powder and water.

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

1.

8. The preparation method according to claim 6, wherein: Adding water to ginseng powder to prepare a fermentation slurry, adding citric acid, and adjusting the pH of the fermentation slurry to 3-5; The enzymolysis agent is added to the fermentation homogenate, and enzymolysis is performed at 53-63° C. for 150-180 minutes, and the pH is adjusted to 4-5 to obtain the ginseng enzymolysis solution.

9. The preparation method according to claim 4, wherein: Keeping the ginseng hydrolysate at 80-90° C. for 40-60 min to obtain a sterilized ginseng hydrolysate; and The sterilized ginseng hydrolysate is cooled to 35-45° C., preferably 35-40° C., the bacterial composition is added thereto, and fermented at 35-45° C., preferably 35-40° C., for 60-80 hours, preferably 65-75 hours, to obtain a ginseng fermentation product.

10. The preparation method according to claim 4, wherein: The ginseng fermentation product comprises 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.

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