A composition having an anemia-improving and anti-fatigue effect and a preparation method thereof

Abstract

The present patent application discloses a composition with the effects of improving anemia and resisting fatigue and a preparation method thereof. The anemia-improving composition of the present application also has the effect of resisting fatigue and comprises a ginseng fermentation product; wherein the ginseng fermentation product is obtained by fermenting a ginseng fermentation medium by using a microbial strain and comprises ginsenoside Rh4, which satisfies the condition that the content of ginsenoside Rh4 is greater than or equal to 7% by weight; and the total amount of ginsenosides in the ginseng fermentation product is 100% by weight. The composition of the present application has the effects of improving anemia and resisting fatigue, has no side effects, has a good taste, and has high safety.

Description

Technical Field

[0001] This invention belongs to the field of biopharmaceutical manufacturing or food biotechnology, specifically relating to a composition rich in rare ginsenosides that has the effects of improving anemia and anti-fatigue, and its preparation method. Background Technology

[0002] Anemia is a common blood disorder characterized by a reduced number of red blood cells in peripheral blood, leading to insufficient oxygen supply to tissues and organs. The global prevalence of anemia is as high as 24.8%, affecting the health of 1.62 billion people. The causes of anemia can be attributed to reduced or insufficient red blood cell production, excessive red blood cell destruction, and blood loss, and may be influenced by dietary habits, long-term exposure to large amounts of chemicals, viral infections, chronic diseases, or malignant tumors. In anemia, the number of red blood cells or the hemoglobin content in the blood decreases, resulting in a reduced oxygen-carrying capacity. Various organs and tissues in the body do not receive sufficient oxygen, especially those with high oxygen demands such as the brain, heart, and muscles. Brain hypoxia can cause symptoms such as dizziness, weakness, and poor concentration, leading to fatigue; the heart beats faster to compensate for insufficient oxygen, which can also exacerbate fatigue over time; and insufficient oxygen to muscles reduces exercise capacity and makes them prone to fatigue.

[0003] Iron deficiency anemia is a common disease, mainly caused by an imbalance between iron supply and demand and reduced hemoglobin synthesis, accounting for 60% to 70% of anemia cases. Simple iron supplementation can easily lead to adverse reactions. Traditional Chinese medicine often uses herbs such as ginseng, codonopsis, astragalus, eleutherococcus senticosus, jujube, placenta, and longan to replenish qi and blood, which is effective and has no side effects.

[0004] Ginseng possesses the effects of greatly replenishing vital energy, strengthening the spleen and lungs, and restoring pulse and preventing collapse. Its active ingredients, including saponins, polysaccharides, amino acids, volatile oils, polyacetylenes, and various other trace elements, are the important material basis for its medicinal effects. Different ginseng components have different properties and pharmacological activities. The main active ingredient in ginseng is ginsenoside. Research has found that secondary metabolic derivatives of ginsenosides, after transformation, have stronger biological activity. These secondary metabolic derivatives are named "rare ginsenosides" because they are scarce in natural ginseng. In contrast, ginsenosides extracted directly from Araliaceae plants are called "prototype ginsenosides."

[0005] By using probiotics to ferment ginseng as a substrate, ginseng fermentation products such as fermented ginseng powder can be obtained. This fermentation process can convert the original ginsenosides into rare ginsenosides, thereby improving their bioactivity and utilization. In addition, ginseng fermentation products have a better taste and can reduce the original bitterness of ginseng.

[0006] According to the literature research conducted by the inventors, ginsenoside Rh4 is the only rare ginsenoside that has been clearly identified as having the effect of improving anemia. However, the content of ginsenoside Rh4 in existing ginseng fermentation products is very low, making it difficult to say that it has a clear and prominent effect of improving anemia. Summary of the Invention

[0007] In view of the aforementioned 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 anemia-improving effects, and a composition using the ginseng fermentation product as a raw material, wherein the composition has a clear and prominent anemia-improving effect. Furthermore, the composition may also have a clear and prominent anti-fatigue effect.

[0008] Through literature review, the inventors discovered that ginsenoside Rh4 is the rare ginsenoside that effectively improves anemia. Based on this, the inventors believed that increasing the content of ginsenoside Rh4 in ginseng fermentation products would allow for the preparation of ginseng fermentation products with enhanced anemia-improving effects. However, a technical challenge is that even among rare ginsenosides, ginsenoside Rh4 is difficult to obtain through microbial fermentation. Its content is very low in the combination of various rare ginsenosides produced by microbial fermentation; if the total ginsenoside content in the ginseng fermentation product is considered as 100% by weight, the content of ginsenoside Rh4 generally does not exceed 1% by weight.

[0009] Furthermore, through in-depth research, the inventors discovered that fermenting a ginseng substrate using a specific combination of *Lactobacillus plantarum* and *Lactobacillus casei* can produce a ginseng fermentation product with a high content of ginsenoside Rh4. Moreover, using this ginseng fermentation product as a raw material, a composition for improving anemia can be prepared. Furthermore, because ginsenoside Rh4 also has anti-fatigue effects, the inventors also discovered that by combining the above-mentioned ginseng fermentation product with invigorating and anti-fatigue components such as honeysuckle powder, jasmine tea powder, B vitamins, and taurine, a composition with both anemia-improving and anti-fatigue effects can be obtained.

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

[0011] 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 following:

[0012] The content of ginsenoside Rh4 is ≥7% by weight, preferably ≥8% by weight;

[0013] The total amount of ginsenosides in the ginseng fermentation product is 100% by weight.

[0014] The ginseng fermentation product can be liquid, solid, or a mixture of solid and liquid, such as fermented ginseng liquid or fermented ginseng powder.

[0015] 2. A microbial composition comprising Lactobacillus plantarum and Lactobacillus casei; wherein,

[0016] The Lactobacillus plantarum mentioned is Lactobacillus plantarum GBFG 61, which is deposited at the China General Microbiological Culture Collection Center (CGMCC) with accession number GGMCC NO: 31583 and deposit date of August 14, 2024.

[0017] 3. The aforementioned microbial composition can be used to ferment ginseng fermentation substrate to produce rare ginsenosides.

[0018] Here, the ginseng fermentation substrate refers to a fermentation substrate containing ginseng fines. The ginseng fines are obtained by refining ginseng, preferably using physical methods such as extrusion, cutting, or grinding. The ginseng fines can be, for example, ginseng powder. The ginseng fermentation substrate may also contain other components typically found in fermentation substrates, such as nitrogen sources, carbon sources, inorganic salts, and pH adjusters.

[0019] 4. The bacterial composition, wherein the dry weight ratio of Lactobacillus plantarum and Lactobacillus casei is 1:3~5.

[0020] 5. The bacterial composition, wherein the Lactobacillus casei is Lactobacillus casei LC-12.

[0021] 6. A method for preparing a ginseng fermentation product rich in ginsenoside Rh4, comprising inoculating a microbial 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;

[0022] The microbial composition is the aforementioned microbial composition.

[0023] 7. The preparation method further includes sterilizing the ginseng fermentation product.

[0024] 8. The preparation method described above, wherein the ginseng fermentation product is the aforementioned ginseng fermentation product.

[0025] 9. The preparation method described above, wherein the ginseng fermentation product is fermented ginseng liquid or fermented ginseng powder.

[0026] 10. A method for manufacturing a composition for improving anemia, comprising:

[0027] The ginseng fermentation product was prepared using the aforementioned method; and

[0028] The ginseng fermentation product is mixed with other components of the anemia-improving composition to obtain the anemia-improving composition.

[0029] 11. A composition for improving anemia, comprising the aforementioned ginseng fermentation product.

[0030] 12. The anemia-improving composition further contains energizing or anti-fatigue ingredients, and also has anti-fatigue effects.

[0031] 13. The anemia-improving composition, wherein the energizing or anti-fatigue component comprises one or more of honeysuckle, jasmine tea powder, B vitamins (e.g., vitamin B1, vitamin B2, vitamin B6) and taurine.

[0032] 14. The anemia-improving composition comprises 15-300 parts by weight of the ginseng fermentation product, 30-54 parts by weight of honeysuckle, 30-54 parts by weight of jasmine tea powder, 0.2-0.25 parts by weight of vitamin B1, 0.1-0.2 parts by weight of vitamin B2, 0.1-0.2 parts by weight of vitamin B6, and 0.05-0.07 parts by weight of taurine.

[0033] 15. The anemia-improving composition is a liquid.

[0034] The anemia-improving composition may also contain one or more of the following: sugars, sugar alcohols, non-sugar and non-sugar alcohol sweeteners, pectin, soluble dietary fiber, salts, other carbohydrates, carbon dioxide, or other food additives. The sugars include one or more of glucose, sucrose, or fructose; the non-sugar and sugar alcohol sweeteners include one or more of stevia, aspartame, sucralose, monk fruit, glycyrrhizin, betaine, or sodium saccharin; the sugar alcohols include one or more of xylitol, erythritol, maltitol, isomaltitol, or sorbitol; the salts include one or more of sodium salts, potassium salts, and phosphates; the other carbohydrates include oligosaccharides, starch, and / or soluble and insoluble dietary fiber; the other food additives include one or more of flavorings, colorings, malic acid, citric acid, sodium citrate, sorbic acid, potassium sorbate, benzoic acid, sodium benzoate, edible gum base, sucrose esters, soybean lecithin, and sodium alginate, wherein the flavorings and colorings are artificially synthesized or natural; and the caffeine is derived from cocoa, coffee, guarana, tea, or chocolate.

[0035] 16. The method for preparing the anemia-improving composition, comprising:

[0036] The ginseng fermentation product was prepared using the aforementioned preparation method; and

[0037] The ginseng fermentation product is mixed with other components of the anemia-improving composition to obtain the anemia-improving composition.

[0038] Beneficial effects of the invention

[0039] Ginseng fermentation products have the effects of replenishing qi and blood and relieving fatigue; honeysuckle contains a certain amount of iron, which can help supplement iron and thus prevent or treat iron-deficiency anemia; jasmine tea has the effects of clearing heat, relieving depression and calming the mind, refreshing the brain and anti-oxidation; vitamins B1, B2, B6 and taurine can all promote iron absorption, increase the hemoglobin content in tissues, and also have anti-fatigue effects. Therefore, the anemia-improving composition of the present invention has a clear and prominent effect of improving anemia and / or relieving fatigue, and has no side effects, tastes good, and is safe to eat, and can be widely used in foods and medicines for improving anemia. Attached Figure Description

[0040] Figure 1 The high-performance liquid chromatogram is used to determine the content of ginsenoside Rh4 in the fermented ginseng powder prepared in Example 1.

[0041] Figure 2 The high-performance liquid chromatogram is used to determine the content of ginsenoside Rh4 in the fermented ginseng powder prepared in Comparative Example 3.

[0042] Figure 3 The figure shows the effect of the anemia-improving and fatigue-relieving composition 1 on the duration of weight-bearing swimming in mice. Detailed Implementation

[0043] The present invention will now be described in detail with reference to specific embodiments. Unless otherwise specified, the scientific terms used in this specification have the meanings commonly understood by those skilled in the art; in case of conflict, the definitions in this specification shall prevail.

[0044] Example

[0045] The plant lactobacillus ( Lactobacillus plantarum GBFG 61 is a β-glucosidase-producing lactobacillus strain isolated and cultivated from soil in a ginseng planting base in Fusong County, Baishan City, Jilin Province. This lactobacillus strain is named *Lactobacillus plantarum* GBFG 61 and is deposited at the China General Microbiological Culture Collection Center (CGMCC) with accession number CGMCC No. 31583. The deposit date is August 14, 2024. The address of the depository is: No. 3, Courtyard 1, Beichen West Road, Chaoyang District, Beijing, Institute of Microbiology, Chinese Academy of Sciences; Postcode: 100101.

[0046] The Lactobacillus casei ( Lactobacillus caseiLC-12 was purchased from Shandong Zhongke Jiayi Biotechnology Co., Ltd. Lactobacillus casei LC-G11 was purchased from Runying Biotechnology (Shanghai) Co., Ltd. Lactobacillus plantarum Lp90 was purchased from Weikang Probiotics (Suzhou) Co., Ltd. Ginseng was purchased from a traditional Chinese medicine market; other raw materials and reagents were obtained through commercial channels.

[0047] Example 1: Preparation of fermented ginseng powder rich in rare ginsenoside Rh4

[0048] Includes the following steps:

[0049] (1) Ginseng powder

[0050] Ginseng is crushed, and ginseng powder of 60-80 mesh is selected as the fermentation raw material.

[0051] (2) Preparation of culture medium

[0052] A ginseng-containing fermentation medium was prepared by mixing 40% by weight of ginseng powder, 1% by weight of glucose, 0.3% by weight of soybean protein powder, 0.04% by weight of magnesium sulfate, 0.015% by weight of manganese sulfate and water. The medium was then sterilized at 121°C for 15 min.

[0053] (3) Vaccination

[0054] After the sterilized culture medium temperature drops below 37℃, Lactobacillus plantarum GBFG 61 powder and Lactobacillus casei LC-12 powder are inoculated into the fermentation culture medium at a ratio of 1:3 and an inoculation amount of 3% by weight.

[0055] (4) Fermentation: Place the inoculated fermentation broth in an incubator at a temperature of 37°C for 22 hours.

[0056] (5) Post-processing: The fermented ginseng liquid was sterilized at 115℃ for 20 minutes. The sterilized fermented liquid was centrifuged at 4500 rpm for 30 minutes to obtain a precipitate. The precipitate was then freeze-dried to obtain fermented ginseng powder.

[0057] Example 2

[0058] The ratio of Lactobacillus plantarum GBFG 61 and Lactobacillus casei LC-12 in Example 1 was changed to 1:4, while all other conditions remained unchanged.

[0059] Example 3

[0060] The ratio of Lactobacillus plantarum GBFG 61 and Lactobacillus casei LC-12 in Example 1 was changed to 1:5, while all other conditions remained unchanged.

[0061] Comparative Example 1

[0062] In Example 1, only Lactobacillus plantarum GBFG 61 was inoculated, while all other conditions remained unchanged.

[0063] Comparative Example 2

[0064] In Example 1, only Lactobacillus casei LC-12 was inoculated, while all other conditions remained unchanged.

[0065] Comparative Example 3

[0066] Replace Lactobacillus plantarum GBFG 61 in Example 1 with Lactobacillus plantarum Lp90, while keeping all other conditions unchanged.

[0067] Comparative Example 4

[0068] Replace Lactobacillus casei LC-12 in Example 1 with Lactobacillus casei LC-G11, while keeping all other conditions unchanged.

[0069] Example 5: Determination of the content of rare ginsenoside Rh4

[0070] Fermented ginseng powders prepared in Examples 1-3 and Comparative Examples 1-4 were dissolved in methanol to prepare solutions of 80 mg / mL. 1 mL of each solution was filtered through a 0.45 μm microporous membrane to obtain a clear liquid of fermented ginseng powder. The total ginsenoside content and the content of various rare ginsenosides were then determined. The content of ginsenoside Rh4 (wt%) was calculated, with the total ginsenoside content as 100 wt%. The results are shown in Table 1.

[0071] The determination method for total ginsenosides was performed according to the method for content determination under the ginsenosides section of Part I of the 2020 edition of the Pharmacopoeia of the People's Republic of China. The results are expressed as ginsenoside Re(C 48 H 82 O 18 )count.

[0072] The detection method for rare ginsenosides shall be in accordance with the method for determining the content of rare ginsenosides in T / CNHFA 001-2021.

[0073] As an example, the high-performance liquid chromatograms for determining the ginsenoside Rh4 content in the fermented ginseng powder prepared in Example 1 and Comparative Example 3 are shown below. Figure 1 and Figure 2 As shown.

[0074] Table 1. Content of rare ginsenoside Rh4 in fermented ginseng powder

[0075]

[0076] As shown in Table 1, when the fermentation strain combination of *Lactobacillus plantarum* GBFG 61 and *Lactobacillus casei* LC-12 of this invention was selected, the content of rare ginsenoside Rh4 in the resulting fermented ginseng powder was significantly increased. This suggests that the resulting fermented ginseng powder has an enhanced effect in improving anemia. It should also be noted that, in addition to *Lactobacillus plantarum* Lp90 and *Lactobacillus casei* LC-G11 as compared in the above examples, the inventors tried various self-isolated or commercially purchased *Lactobacillus plantarum*, *Lactobacillus casei*, and combinations thereof, but none could achieve the technical effect of a rare ginsenoside Rh4 content of 7% by weight or higher.

[0077] Example 6: Effects of different fermented ginseng powders on rats with iron deficiency anemia

[0078] Forty healthy weaned rats were selected and allowed to adapt to the experimental environment for 3 days. They were then randomly divided into a normal control group (n=10) and a low-iron experimental group (n=30). The normal control group was fed normal feed and deionized water, while the low-iron experimental group was fed low-iron feed and deionized water. At the same time, the rats were bled by tail clipping once every 5 days, with 0.4 ml of blood each time. Starting from week 3, tail blood was collected from a portion of rats weekly to measure hemoglobin (Hb). Rats with an Hb concentration of approximately 60 g / L were selected as experimental animals. Based on the Hb level and body weight of the anemic rats, they were randomly divided into a low-iron model group and two experimental groups. All groups continued to be fed a low-iron diet, while 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 samples, abdominal aortic blood was collected from the rats, and hemoglobin, red blood cells (RBC), and hematocrit (HCT) were detected using a fully automated blood analyzer. The test results are shown in Table 2.

[0079] Table 2. Effects of different dosage groups on hemoglobin and erythrocyte count in rats

[0080]

[0081] Note: Compared with the normal group, *** P <0.001; compared with the model group, ### P <0.001.

[0082] As shown in Table 2, in Example 1 group, after treatment, the levels of Hb, RBC, and HCT in anemic rats were significantly restored, showing a significant difference compared to the model group and approaching the levels of the normal group, indicating that it has a significant effect in improving anemia. In contrast, in Comparative Example 3 group, although the levels of Hb, RBC, and HCT in anemic rats were restored, the difference was not significant compared to the model group, but significant compared to the normal group, indicating that its effect in improving anemia was limited.

[0083] Example 7: Preparation of a Composition for Improving Anemia and Combating Fatigue

[0084] Weigh the following components by weight: 30 parts fermented ginseng powder, 40 parts honeysuckle, 36 parts jasmine tea powder, 0.225 parts vitamin B1, 0.15 parts vitamin B2, 0.15 parts vitamin B6, and 0.065 parts taurine. Mix the above ingredients thoroughly, then add 1500 parts erythritol, 60 parts citric acid, and a certain amount of water. Stir well, then aseptically fill into containers to obtain Composition 1 for improving anemia and combating fatigue.

[0085] Example 8: Verification of the anemia-improving and anti-fatigue composition's efficacy in improving anemia.

[0086] The efficacy of the anemia-improving and fatigue-relieving composition 1 was verified. The experimental procedure was the same as in Example 6. The dosage of the anemia-improving and fatigue-relieving composition 1 was 80 mg / (kg BW). Based on the effective components in the anemia-improving and fatigue-relieving composition 1, it does not contain erythritol, citric acid, or water. The results of hemoglobin and red blood cell counts, as well as hematocrit, are shown in Table 3.

[0087] Table 3. Effects of the drug-administered composition on hemoglobin and erythrocyte count, and hematocrit in rats.

[0088]

[0089] Note: Compared with the normal group, *** P <0.001; compared with the model group, ### P <0.001.

[0090] As shown in Table 3, after treatment with the anemia-improving and fatigue-relieving composition of the present invention, the levels of Hb, RBC, and HCT in anemic rats were significantly restored, showing significant differences compared with the model group and no significant differences compared with the normal group, indicating that it has a good effect on improving anemia.

[0091] Example 9: Verification of the anti-fatigue efficacy of the composition for improving anemia and combating fatigue

[0092] The anti-fatigue efficacy of the above-mentioned anemia-improving and anti-fatigue composition 1 was verified.

[0093] Twenty SPF-grade male Kunming mice weighing 20-25g were acclimatized for 7 days. After daily swimming training, they were randomly divided into two groups: the anemia-improving and anti-fatigue composition group 1 and the control group, with 10 mice in each group. In addition to normal daily feeding, the mice in each group were administered 80 mg / (kg BW) of the anemia-improving and anti-fatigue composition 1 and an equal volume of physiological saline by gavage for 30 days. Thirty minutes after the last administration, the mice in each group were made to swim with a 5% lead weight, and the swimming time was recorded. The results of the weight-bearing swimming time test are as follows: Figure 3 As shown.

[0094] Depend on Figure 3 It can be seen that the anemia-improving and fatigue-relieving composition of the present invention has good anti-fatigue effects (compared with the model group, ##P<0.01).

[0095] The above embodiments are preferred embodiments of the present invention, but the embodiments of the present invention are not limited to the above embodiments. For those skilled in the art, other variations or modifications can be made without departing from the principles of the present invention, and the resulting variations are still within the scope of protection of the present invention.

[0096] Industrial applicability

[0097] This invention provides a ginseng fermentation product containing rare ginsenosides with enhanced anemia-improving effects, its preparation method, and its application.

Claims

1. An anemia improving composition comprising a ginseng fermentation product; wherein, The ginseng fermentation product is obtained by fermenting a ginseng fermentation substrate using a microbial strain, and contains ginsenoside Rh4, which satisfies: The content of ginsenoside Rh4 is ≥ 7% by weight, based on the total amount of ginsenosides in the ginseng fermentation product; The microbial strain is a strain composition consisting of Lactobacillus plantarum and Lactobacillus casei; wherein, The Lactobacillus plantarum is Lactobacillus plantarum GBFG 61, which is preserved in the China General Microbiological Culture Collection Center on August 14, 2024, with the preservation number GGMCC NO: 31583; The Lactobacillus casei is Lactobacillus casei LC-12; The dry weight ratio of the Lactobacillus plantarum and the Lactobacillus casei is 1:3-5.

2. The anemia-improving composition of claim 1, which also has an anti-fatigue effect, and further comprises a refreshing or anti-fatigue ingredient.

3. The anemia improving composition according to claim 2, wherein The refreshing or anti-fatigue ingredient includes one or more or all selected from honeysuckle, jasmine tea powder, B vitamins, and taurine.

4. The anemia-improving composition of claim 1 or 2, which comprises 15-300 parts by weight of the ginseng fermentation product, 30-54 parts by weight of honeysuckle, 30-54 parts by weight of jasmine tea powder, 0.2-0.25 parts by weight of vitamin B1, 0.1-0.2 parts by weight of vitamin B2, 0.1-0.2 parts by weight of vitamin B6, and 0.05-0.07 parts by weight of taurine.

5. The anemia-improving composition of claim 1 or 2, which is in a liquid or solid form.

6. The anemia-improving composition of claim 1 or 2, which satisfies: the content of ginsenoside Rh4 is ≥ 8% by weight, based on the total amount of ginsenosides in the ginseng fermentation product.

7. A method for preparing the anemia-improving composition of claim 1, which comprises: inoculating a strain composition into a ginseng fermentation substrate and fermenting at 37°C for 20-30 hours, thereby obtaining a ginseng fermentation product containing rare ginsenoside Rh4; and mixing the ginseng fermentation product with other components of the anemia-improving composition, thereby obtaining the anemia-improving composition; wherein, the strain composition consists of Lactobacillus plantarum and Lactobacillus casei; the Lactobacillus plantarum is Lactobacillus plantarum GBFG 61, which is preserved in the China General Microbiological Culture Collection Center on August 14, 2024, with the preservation number GGMCC NO: 31583; the Lactobacillus casei is Lactobacillus casei LC-12; the dry weight ratio of the Lactobacillus plantarum and the Lactobacillus casei is 1:3-5.

8. The production method according to claim 7, wherein The ginseng fermentation product is a fermented ginseng liquid or a fermented ginseng powder.

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