Ginseng fermentation product rich in ginsenoside Rh4 as well as preparation method and application thereof
By combining Lactobacillus plantarum and Lactobacillus casei fermentation of the ginseng matrix, the content of ginseng saponin Rh4 was improved, the problem of low Rh4 content in microbial fermentation was solved, and the effect of improving anemia was significantly improved.
Patent Information
- Application Number
- CN202411582420.5
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2024-11-07
- Publication Date
- 2025-01-07
AI Technical Summary
The existing microbial fermentation method is difficult to efficiently produce ginseng saponin rich in Rh4, which leads to its low content in ginseng saponin compositions and the inability to clearly improve the anemia effect.
The ginseng matrix is fermented with a combination of Lactobacillus plantarum and Lactobacillus casei. By controlling the fermentation conditions, the content of ginseng saponin Rh4 is increased to ≥7%, forming a ginseng fermentation product rich in Rh4.
The content of ginseng saponin Rh4 in the ginseng fermentation products was significantly improved, and the medicinal effect of improving anemia was enhanced. It was also verified through experiments that it had a significant improvement effect on rats with iron deficiency anemia.
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Abstract
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 rare ginsenoside Rh4 with enhanced anemia-improving effect, and a biological fermentation preparation method and application thereof. Background Art
[0002] Ginseng is the dried root and rhizome of Panax ginseng CAMey. It has extremely high medicinal and edible value and is rich in a variety of active ingredients. Ginseng has the effects of replenishing vital energy, strengthening the spleen and lungs, and restoring the pulse and consolidating the body. The active ingredients it contains, such as saponins, polysaccharides, amino acids, volatile oils, polyacetylenes, and a variety of other trace elements, are important material bases for its efficacy. The properties and pharmacological activities of different ginseng components are also different. The main active ingredient of ginseng is ginsenoside. Studies have found that the secondary metabolite derivatives of ginsenosides after transformation have stronger biological activity. This secondary metabolite derivative is named "rare ginsenoside" because of its rare content in natural ginseng. In contrast, ginsenosides directly extracted from Araliaceae plants are called "prototype ginsenosides". Rare saponins are particularly effective in anti-tumor, regulating the body's immunity, protecting the nervous system, etc., and have higher bioavailability, so they have extremely high medicinal development value and broad application space. 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 improving immunity.
[0003] 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.
[0004] 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 ginsenoside 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.
[0005] On the other hand, according to the literature research conducted by the inventor, ginsenoside Rh4 is the only rare ginsenoside that has a clear effect on improving anemia, but its content in natural Araliaceae Panax genus plants is extremely low, only at the ppm level; moreover, ginsenoside Rh4 is also difficult to obtain through microbial fermentation and its content in the composition of various rare ginsenosides produced by microbial fermentation is very low. Therefore, it is difficult to say that the existing ginsenoside composition has a clear and outstanding effect on improving anemia. 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 ginsenoside Rh4 with enhanced efficacy in improving anemia, and a preparation method and application thereof.
[0007] After literature research, the inventors found that among the rare ginsenosides, ginsenoside Rh4 is the one that can improve anemia. Based on this, the inventors believe that if the content of ginsenoside Rh4 in ginseng fermentation products can be increased, ginseng fermentation products with enhanced anemia-improving effects can be prepared. However, the technical difficulty is that even among the rare ginsenosides, ginsenoside Rh4 is difficult to obtain through microbial fermentation conversion, and its content in the composition of various rare ginsenosides produced by microbial fermentation is very low. If the total amount of ginsenosides in ginseng fermentation products is 100% by weight, the content of ginsenoside Rh4 generally does not exceed 1% by weight.
[0008] Furthermore, after in-depth research, the inventors found that fermenting a ginseng matrix with a specific combination of Lactobacillus plantarum and Lactobacillus casei can produce a ginseng fermentation product with a high content of ginsenoside Rh4, thereby completing the present invention.
[0009] The present invention comprises:
[0010] 1. A ginseng fermentation product rich in ginsenoside Rh4, obtained by fermenting a ginseng fermentation substrate using a microbial strain, comprising ginsenoside Rh4, and satisfying:
[0011] The content of ginsenoside Rh4 is ≥7 wt%, preferably ≥8 wt%;
[0012] The total amount of ginsenosides in the ginseng fermentation product is taken as 100% by weight.
[0013] The ginseng fermentation product may be liquid or solid or a solid-liquid mixture, for example, fermented ginseng liquid or fermented ginseng powder.
[0014] 2. A bacterial composition, which consists of Lactobacillus plantarum and Lactobacillus casei; wherein:
[0015] The plant lactobacillus is plant lactobacillus GBFG 61, which is deposited in the General Microbiology Center of China National Microbiological Culture Collection Administration, with a deposit number of GGMCC NO: 31583 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 Lactobacillus plantarum to Lactobacillus casei is 1:3-5.
[0019] 5. The bacterial composition according to item 2, wherein the Lactobacillus casei is Lactobacillus casei LC-12.
[0020] 6. A method for preparing a ginseng fermentation product rich in ginsenoside Rh4, comprising inoculating a bacterial composition into a ginseng fermentation substrate, and fermenting at 36-39°C, preferably 37°C, for 10-50 hours, preferably 15-40 hours, more preferably 20-30 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 fermented ginseng liquid or fermented ginseng powder.
[0025] 10. A ginseng fermentation product obtained by the preparation method of any one of items 6 to 9. BRIEF DESCRIPTION OF THE DRAWINGS
[0026] Figure 1 This is a high performance liquid chromatogram for determining the content of ginsenoside Rh4 in the fermented ginseng powder prepared in Example 1.
[0027] Figure 2 This is a high performance liquid chromatogram of the determination of the content of ginsenoside Rh4 in the fermented ginseng powder prepared in Comparative Example 3. DETAILED DESCRIPTION
[0028] 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.
[0029] Example
[0030] The plant lactobacillus GBFG 61 is a lactobacillus strain that produces β-glucosidase isolated and cultivated from the soil of the ginseng planting base in Fusong County, Baishan City, Jilin Province. The lactobacillus strain was named plant lactobacillus (Lactobacillus plantarum) GBFG 61, which is deposited in the General Microbiological Center of the China Microbiological Culture Collection Administration Committee, and the deposit number is CGMCC No.31583. The deposit date is August 14, 2024, and the deposit address is: Institute of Microbiology, Chinese Academy of Sciences, No. 3, No. 1, Beichen West Road, Chaoyang District, Beijing; Postal code of the deposit agency: 100101.
[0031] The Lactobacillus casei LC-12 was purchased from Shandong Zhongke Jiayi Bioengineering Co., Ltd. The Lactobacillus casei LC-G11 was purchased from Runying Bioengineering (Shanghai) Co., Ltd. The Lactobacillus plantarum Lp90 was purchased from Weikang Probiotics (Suzhou) Co., Ltd. Ginseng was purchased from the Chinese herbal medicine market, and other raw materials and reagents were purchased from commercial channels.
[0032] Example 1 Preparation of fermented ginseng powder rich in rare ginsenoside Rh4
[0033] The steps include:
[0034] (1) Grinding of ginseng
[0035] Ginseng was crushed and ginseng powder with mesh size of 60-80 was selected as fermentation raw material.
[0036] (2) Preparation of culture medium
[0037] 40 wt% of ginseng powder, 1 wt% of glucose, 0.3 wt% of soy protein powder, 0.04 wt% of magnesium sulfate, 0.015 wt% of manganese sulfate and water were uniformly mixed to prepare a fermentation medium containing ginseng, which was sterilized at 121° C. for 15 min.
[0038] (3) Vaccination
[0039] After the temperature of the sterilized culture medium drops below 37° C., Lactobacillus plantarum GBFG 61 powder and Lactobacillus casei LC-12 powder are inoculated into the fermentation medium at a ratio of 1:3 and an inoculation amount of 3% by weight.
[0040] (4) Fermentation: The fermented liquid after inoculation is placed in an incubator at a fermentation temperature of 37° C. for 22 hours.
[0041] (5) Post-treatment: The fermented ginseng liquid was sterilized at 115°C for 20 min. The sterilized fermented liquid was centrifuged at 4500 rpm for 30 min to obtain a precipitate, which was freeze-dried to obtain fermented ginseng powder.
[0042] Example 2
[0043] The ratio of Lactobacillus plantarum GBFG 61 to Lactobacillus casei LC-12 in Example 1 was changed to 1:4, and the other conditions remained unchanged.
[0044] Example 3
[0045] The ratio of Lactobacillus plantarum GBFG 61 to Lactobacillus casei LC-12 in Example 1 was changed to 1:5, and the other conditions remained unchanged.
[0046] Comparative Example 1
[0047] In Example 1, only Lactobacillus plantarum GBFG 61 was inoculated, and the other conditions remained unchanged.
[0048] Comparative Example 2
[0049] Only Lactobacillus casei LC-12 was inoculated in Example 1, and the other conditions remained unchanged.
[0050] Comparative Example 3
[0051] The Lactobacillus plantarum GBFG 61 in Example 1 was replaced with Lactobacillus plantarum Lp90, and the other conditions remained unchanged.
[0052] Comparative Example 4
[0053] The Lactobacillus casei LC-12 in Example 1 was replaced by Lactobacillus casei LC-G11, and the other conditions remained unchanged.
[0054] Example 5 Determination of the content of rare ginsenoside Rh4
[0055] The fermented ginseng powders prepared in Examples 1-3 and Comparative Examples 1-4 were respectively dissolved in methanol and prepared into 80 mg / mL. 1 mL of the solution was respectively taken and filtered with a 0.45 μm microporous filter membrane to obtain the fermented ginseng powder clear solution. The total ginsenoside content and the content of various rare ginsenosides therein were determined, and the ginsenoside Rh4 content (wt %, with the total ginsenosides as 100 wt %) was calculated. The results are shown in Table 1.
[0056] The detection method of total ginsenosides 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.
[0057] 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.
[0058] As an example, the HPLC chromatograms for determining the content of ginsenoside Rh4 in the fermented ginseng powder prepared in Example 1 and Comparative Example 3 are as follows: Figure 1 and Figure 2 shown.
[0059] Table 1. Content of rare ginsenoside Rh4 in fermented ginseng powder
[0060]
[0061]
[0062] As can be seen from Table 1, when the plant lactobacillus GBFG 61 strain of the present invention and the lactobacillus casei LC-12 strain fermentation strain combination are selected, the content of rare ginsenoside Rh4 in the obtained fermented ginseng powder is significantly improved. This prompts that the obtained fermented ginseng powder has a strengthened effect of improving anemia. It should also be noted that, in addition to the plant lactobacillus Lp90 and the lactobacillus casei LC-G11 of the above-mentioned example comparison, the inventor has tried a variety of plant lactobacillus, lactobacillus casei and combinations thereof that are separated by themselves or purchased commercially, and all cannot achieve the technical effect that the content of rare ginsenoside Rh4 is more than 7% by weight.
[0063] Example 6 Effects of different fermented ginseng powders on iron deficiency anemia rats
[0064] Forty healthy weaned rats were selected and adapted to the experimental environment for 3 days, and then randomly divided into a normal control group of 10 rats and a low-iron experimental group of 30 rats. The normal group was fed with normal feed and deionized water, and the low-iron experimental group was fed with low-iron feed and deionized water. At the same time, blood was collected by tail-cutting method, once every 5 days, 0.4 ml each time. Starting from the third week, some rats were selected every week to collect tail blood for hemoglobin (Hb) measurement. Rats with an Hb mass concentration of about 60 g / L were selected as experimental animals. According to the Hb level and body weight of anemic rats, they were randomly divided into a low iron model group and two experimental groups. Each group continued to be fed with a low iron feed, and the low iron control group was given the corresponding solvent. The two experimental groups were the fermented ginseng powder group (50 mg / kg BW) in Example 1 and the fermented ginseng powder group (50 mg / kg BW) in Comparative Example 3. After 30 days of administration of the test sample, the abdominal aorta blood of the rats was taken, and the hemoglobin, red blood cell (RBC), and hematocrit (HCT) were detected using a fully automatic blood analyzer. The test results are shown in Table 2.
[0065] Table 2. Effects of each dose group on hemoglobin and red blood cell count in rats
[0066]
[0067] Note: Compared with the normal group, ***P<0.001; compared with the model group, ### P<0.001.
[0068] As shown in Table 2, as for the Example 1 group, after treatment, the Hb, RBC, and HCT levels of the anemic rats were significantly restored, with significant differences compared with the model group, and were close to the levels of the normal group, indicating that it has a significant effect on improving anemia; on the contrary, as for the Comparative Example 3 group, although the Hb, RBC, and HCT levels of the anemic rats were restored, the difference was not significant compared with the model group, but was significant compared with the normal group, indicating that its effect on improving anemia is limited.
[0069] The above embodiments are preferred implementations of the present invention, but the implementations of the present invention are not limited to the above embodiments. For ordinary technicians in this technical field, other different forms of changes or modifications can be made without departing from the principles of the present invention. The derived changes are still within the scope of the creative protection of the present invention.
[0070] Industrial Applicability
[0071] The present invention provides a ginseng fermentation product containing rare ginsenoside Rh4 and having enhanced anemia-improving effect, and a preparation method and application thereof.
Claims
1. A ginseng fermentation product rich in ginsenoside Rh4, obtained by fermenting a ginseng fermentation substrate using a microbial strain, comprising ginsenoside Rh4, and satisfying: The content of ginsenoside Rh4 is ≥7 wt%, preferably ≥8 wt%; The total amount of ginsenosides in the ginseng fermentation product is taken as 100% by weight.
2. A bacterial composition, which consists of Lactobacillus plantarum and Lactobacillus casei; wherein: The plant lactobacillus is Lactobacillus plantarum GBFG 61, which is deposited in the General Microbiological Center of China National Microbiological Culture Collection Administration, with a deposit number of GGMCCNO: 31583 and a deposit date of August 14, 2024; and The Lactobacillus casei is Lactobacillus casei LC-12.
3. The bacterial composition according to claim 2, which can be used to ferment ginseng fermentation matrix to produce rare ginsenosides.
4. The bacterial composition according to claim 2, wherein The dry weight ratio of the Lactobacillus plantarum to the Lactobacillus casei is 1:3-5.
5. A method for preparing a ginseng fermentation product rich in ginsenoside Rh4, comprising inoculating a bacterial composition into a ginseng fermentation substrate, and fermenting at 36-39° C. for 10-50 hours, preferably 15-40 hours, more preferably 20-30 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 to 4. The preparation method according to claim 5 , further comprising sterilizing the ginseng fermentation product.
7. The preparation method according to claim 5, wherein: The ginseng fermentation product is the ginseng fermentation product according to claim 1.
8. The preparation method according to claim 5, wherein The ginseng fermentation product is fermented ginseng liquid or fermented ginseng powder.