A ginsenoside probiotic composition and applications thereof

Abstract

This invention belongs to the field of biotechnology, specifically relating to the preparation, conversion, and application of a ginsenoside probiotic composition. By adding probiotics capable of degrading ginsenosides to ginseng products, this invention enhances the bioavailability of ginsenosides in the human body by degrading ginsenosides into ginsenoside diol and ginsenoside triol. The core aglycone of this probiotic composition achieves a conversion rate exceeding 80% within one hour, providing better and more convenient treatment options in anti-inflammatory, immune-regulating, and other related fields, thereby increasing therapeutic efficacy.

Description

Technical Field

[0001] This invention belongs to the field of biotechnology, specifically relating to the preparation of a ginsenoside probiotic composition, the conversion of ginsenosides, and their applications. Background Technology

[0002] Ginseng is a top-grade traditional Chinese medicine for tonifying the body. The "Four Hundred Herbs of Traditional Chinese Medicine" describes ginseng as having a sweet taste, greatly replenishing vital energy, relieving cough and promoting body fluid production, and regulating nutrition and defense. Modern pharmacological research shows that ginsenosides are the main active components of ginseng. Ginsenosides possess antioxidant, anti-inflammatory, antibacterial, cardiovascular disease-fighting, anti-diabetic, anti-cancer, obesity-reducing, and energy-enhancing properties. Currently, over 40 monomeric saponin components have been identified in ginseng, with ginsenosides Ra1, Rb1, Rb2, Rc, Rd, Re, Rf, and Rg1 accounting for over 95% of the content. These high-content ginsenosides can be hydrolyzed to yield rare ginsenosides such as Rg3, Rh1, Rh2, and CK. These rare ginsenosides exhibit good pharmacological activity against the nervous, cardiovascular, endocrine, and digestive systems, showing great promise for future applications. In the field of adjuvant therapy for tumors, some high-purity rare ginsenoside preparations (such as Rg3) have been used clinically as prescription drugs or advanced nutritional supplements to improve patients' quality of life and reduce the side effects of radiotherapy and chemotherapy. In the high-end beauty and skincare field, rare ginsenosides, due to their excellent antioxidant and cell repair capabilities, are regarded as core active ingredients and added to top-tier skincare products to meet the market's high-end demand for anti-aging and repair. In addition, in the field of health and wellness, the industry is using biotechnology such as probiotic fermentation and enzymatic transformation to develop new functional foods rich in rare ginsenosides (such as fermented ginseng drinks and concentrated liquids), promoting the upgrading of traditional ginseng products towards higher added value and responding to modern consumers' pursuit of "medicine and food from the same source" and precise health preservation.

[0003] Probiotics are a class of beneficial microorganisms that are widely present in the human gut and reproductive system. They can regulate the function of the host's mucosa and immune system and improve the balance of the gut microbiota. There are many types of probiotics, and those used in the food industry include bacteria such as Bifidobacterium, Lactobacillus, and Bacillus, as well as fungi such as Saccharomyces cerevisiae, Monascus purpureus, and Ganoderma lucidum. In recent years, there has been considerable research on the fermentation of ginseng using probiotics. Utilizing probiotic fermentation to transform saponins in ginseng is not only highly specific and yield-efficient but also relatively safe, making it an important approach to improving the bioactivity of ginseng products.

[0004] The method of preparing rare ginsenosides through microbial fermentation has developed rapidly in recent years. While some fungi and non-edible microorganisms have a strong ability to convert ginsenosides, safety issues hinder their widespread application. Patent CN113136358B discloses an aerobic co-culture probiotic fermentation process to increase ginsenoside yield; however, this fermentation requires high-yield cellulase-producing Bacillus subtilis, which needs to be cultured and preserved before use, is unstable, and consumes limited resources. Another example is patent CN113897304A, which discloses a composite bacterial strain composition and its application to improve ginsenoside conversion. This composition includes Bifidobacterium bifidum, Lactobacillus acidophilus, Lactobacillus plantarum, and Lactobacillus helveticus, significantly improving the bioavailability of rare ginsenosides and thus effectively increasing their absorption rate in the intestines. However, this probiotic composition has multiple components, requiring the combined use of various probiotics for effectiveness, resulting in high costs, and the probiotics need to be taken long-term to stabilize the bacterial flora.

[0005] Most commercially available products add ginsenosides to probiotics to enhance their effects, or add specific probiotics to ginsenoside extracts to degrade the ginsenosides. However, these methods primarily aim to obtain high-purity rare ginsenosides or core aglycones through in vitro fermentation, rather than to degrade ginsenosides in the body. Therefore, we aim to provide a directly edible probiotic composition that improves the bioavailability of ginsenosides. This composition should produce effects with a single dose, offering better and more convenient treatment options in anti-inflammatory and immune-regulating fields, thereby enhancing therapeutic efficacy. Summary of the Invention

[0006] This invention improves the bioavailability of ginsenosides in the human body by adding probiotics that can degrade ginsenosides into ginsenoside diol and ginsenoside triol.

[0007] In a first aspect, the present invention provides a probiotic composition comprising Bifidobacterium and Lactobacillus.

[0008] Furthermore, the ratio of Bifidobacterium to Lactobacillus in the probiotic composition is 1~10:1~6.

[0009] Furthermore, the ratio of Bifidobacterium to Lactobacillus in the probiotic composition is preferably 1:1.

[0010] Furthermore, the Bifidobacterium is selected from any one of Bifidobacterium adolescentis, Bifidobacterium animalis, lactic acid bacteria, Bifidobacterium, Bifidobacterium difformis, Bifidobacterium infantis, Bifidobacterium longum, Bifidobacterium animalis, and Bifidobacterium lactis.

[0011] Furthermore, the lactobacillus is selected from any one of Lactobacillus plantarum, Lactobacillus casei, lactic acid bacteria, Lactobacillus acidophilus, Lactobacillus helveticus, Lactobacillus johnsonii, Lactobacillus acidophilus, Lactobacillus gasseri, Lactobacillus rhamnosus, and Lactobacillus delbrueckii.

[0012] Furthermore, the Bifidobacterium is selected from Bifidobacterium longum, Bifidobacterium bifidum, and Bifidobacterium animalis; preferably Bifidobacterium longum.

[0013] Furthermore, the lactobacillus is selected from Lactobacillus acidophilus, Lactobacillus rhamnosus, and Lactobacillus plantarum; preferably Lactobacillus plantarum.

[0014] Secondly, the present invention provides a ginsenoside probiotic composition, which is composed of ginseng products and a probiotic composition. The probiotic composition contains at least one Bifidobacterium and one Lactobacillus. After the ginsenoside probiotic composition is taken, the probiotics can degrade ginsenosides into ginsenoside core aglycones during human digestion, thereby improving the bioavailability of ginsenosides.

[0015] Furthermore, the conversion rate of the core aglycone in the ginsenoside probiotic composition exceeds 80% within one hour.

[0016] Furthermore, the conversion rate of the core aglycone in the ginsenoside probiotic composition exceeds 98% within one hour.

[0017] Furthermore, the added ginseng product is selected from one of the following: ginseng slices, ginseng extract, ginseng powder, red ginseng slices, and American ginseng slices.

[0018] Furthermore, the ratio of the ginseng product and the probiotic composition is 1:1 to 100.

[0019] Furthermore, the preferred ratio of the ginseng product and the probiotic composition is 1:10.

[0020] Furthermore, the ratio of Bifidobacterium to Lactobacillus in the probiotic composition is 1~10:1~6.

[0021] Furthermore, the ratio of Bifidobacterium to Lactobacillus in the probiotic composition is preferably 1:1.

[0022] Furthermore, the Bifidobacterium is selected from any one of Bifidobacterium adolescentis, Bifidobacterium animalis, lactic acid bacteria, Bifidobacterium, Bifidobacterium difformis, Bifidobacterium infantis, Bifidobacterium longum, Bifidobacterium animalis, and Bifidobacterium lactis.

[0023] Furthermore, the lactobacillus is selected from any one of Lactobacillus plantarum, Lactobacillus casei, lactic acid bacteria, Lactobacillus acidophilus, Lactobacillus helveticus, Lactobacillus johnsonii, Lactobacillus acidophilus, Lactobacillus gasseri, Lactobacillus rhamnosus, and Lactobacillus delbrueckii.

[0024] Furthermore, the Bifidobacterium is selected from Bifidobacterium longum, Bifidobacterium bifidum, and Bifidobacterium animalis; preferably Bifidobacterium longum.

[0025] Furthermore, the lactobacillus is selected from Lactobacillus acidophilus, Lactobacillus rhamnosus, and Lactobacillus plantarum; preferably Lactobacillus plantarum.

[0026] Thirdly, the present invention provides the use of the probiotic composition as described in the first aspect in any of the following aspects; (1) Preparation of microspheres loaded with ginsenoside probiotic composition; (2) Preparation of nanomedicine delivery systems or devices; (3) Preparation of culture medium for in vitro conversion of ginsenosides; (4) Prepare other health products / dietary supplements.

[0027] Fourthly, the present invention provides a ginsenoside probiotic preparation with anti-inflammatory and immunomodulatory effects, characterized in that the preparation comprises the ginsenoside probiotic composition as described in the second aspect.

[0028] Furthermore, the ginsenoside probiotic composition can be prepared into dosage forms suitable for different populations for treatment.

[0029] Furthermore, the dosage form is selected from one or more of the following: sugar-coated tablets, film-coated tablets, enteric-coated tablets, enteric-coated capsules, capsules, hard capsules, soft capsules, oral liquids, lozenges, granules, powders, pills, boluses, suspensions, powders, tinctures, preparations, drops, injections, powder for injection, creams, sustained-release agents, and targeted agents.

[0030] Furthermore, the preferred dosage form is enteric-coated tablets, enteric-coated capsules, or solid beverages.

[0031] Furthermore, the ginsenoside probiotic composition also contains other active ingredients, including drugs that can promote anti-inflammatory effects / inhibit bacterial growth.

[0032] This invention simulates the human intestinal environment and selects different ratios of ginseng products and probiotic compositions to test the conversion rate of core aglycones. In the probiotic composition (taking Bifidobacterium and Lactobacillus as an example) with a ratio of 1:1, the conversion rate of core aglycones exceeds 80% in one hour. Based on this result, a complete release experiment was designed again. After optimization, it was determined that the conversion rate of core aglycones is optimal when the ratio of ginseng products to probiotics (by weight) is 1:10. Detailed Implementation

[0033] The technical solutions of the present invention will be clearly and completely described below with reference to the embodiments of the present invention. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those of ordinary skill in the art without creative effort are within the scope of protection of the present invention.

[0034] In this invention, the ginsenoside probiotic composition exerts an anti-inflammatory and immunomodulatory effect through glucocorticoid receptors. The specific mechanism is as follows: Glucocorticoid receptors (GR) regulate inflammation, growth, metabolism, and apoptosis. GR primarily acts as a transcription factor, activating or inhibiting the expression of numerous target genes, including many anti-inflammatory and pro-inflammatory molecular genes.

[0035] Ginsenoside diol and ginsenoside triol can bind to GR to activate its nuclear translocation, selectively inhibit the NF-κB pathway, and block the release of pro-inflammatory factors.

[0036] Example 1: Determining the optimal combination of probiotics The ginseng and ginseng extract used in this invention were purchased from Jilin Yuanling Ginseng Ear Co., Ltd.; the probiotics were purchased from Xi'an Juntian Biotechnology Co., Ltd.

[0037] 1. Explanation of experimental data: The recommended daily intake of probiotics for humans is 10 mg / day. (Experimental design: Probiotic intake is 10 mg / day.) 9 - 10 11 CFU Recommended daily intake of ginseng: 1-5g / 70kg, corresponding to a probiotic dosage of 10g. 9 -50 9 CFU / 70kg The recommended formulation, without affecting human health, is 1g : 10g. 8 5 10 8 10 9 5 10 9 10 101g of CFU probiotics. Ginseng powder generally contains approximately 50-70mg of Rb1+Re and approximately 25-35mg of protopanaxadiol+protopanaxtriol.

[0038] Based on the above data, take 3g of ginseng powder as one weight unit, and 3×10 probiotics per unit. 8 CFU is a unit of weight. In this invention, the ratio of ginseng products to probiotics is a weight ratio. The actual weight of different ginseng products is calculated based on the total saponin content ratio in Table 1.

[0039] The total saponin content ratio is shown in Table 1. Table 1. Proportion of total saponins in different ginseng products 2. Methods and Materials: The intestinal melting device is selected according to the standard parameters listed in the pharmacopoeia, specifically: Temperature control: The temperature of the dissolution medium is typically precisely controlled at 37±0.5°C to simulate human body temperature. The proportion of artificial intestinal fluid is shown in Table 2. Table 2 Standards for Intestinal Melting Devices Anaerobic environment: N2:CO2:H2 = 85:10:5 Sampling: Sampling and analysis at a specific time; Sample preparation: Ginseng products were ground into powder and passed through an 80-mesh sieve; According to the experimental ratio, add the corresponding amount of Bifidobacterium longum-Lactobacillus plantarum mixed bacterial solution; add preheated simulated intestinal fluid to the dissolution bottle to the 1000mL mark, gently shake for 1 minute to disperse the ginseng powder evenly and avoid clumping, and then conduct intestinal simulation.

[0040] 3. Testing Methods The steroidal ring structure of the core aglycone molecule of ginsenosides contains 2-3 hydroxyl groups, and acyl chlorides have strong reactivity, readily undergoing nucleophilic substitution reactions with hydroxyl groups. Based on this chemical characteristic, this invention selects benzoyl chloride as a derivatizing reagent. By introducing a benzoyl functional group into the hydroxyl site of ginsenosides, the target compound acquires ultraviolet absorption capability, providing conditions for subsequent detection and analysis.

[0041] 1) Preparation of standard stock solution Accurately weigh 25.0 mg of ginsenoside diol and 25.0 mg of ginsenoside triol reference standards, place them in a 25 ml volumetric flask, dissolve and dilute to the mark with anhydrous pyridine, and shake well. Prepare a 1.0 mg / ml standard stock solution of ginsenoside diol and ginsenoside triol.

[0042] 2) Preparation of derivatization standard series solutions Accurately pipette 0.1, 0.2, 0.4, 0.6, 0.8, and 1.0 ml of 1.0 mg / ml ginsenoside diol and ginsenoside triol standard stock solutions into 25 ml reaction flasks. Add 3.0 times the volume of benzoyl chloride reagent and 2.0 times the volume of anhydrous pyridine, respectively. Reflux in an 80°C water bath for 2 hours, then remove and dry under reduced pressure with a nitrogen stream. Dissolve in methanol and transfer to a 10 ml volumetric flask, dilute to the mark, and mix well. This yields a series of ginsenoside diol and ginsenoside triol derivatization standard solutions with concentrations of 0.01, 0.02, 0.04, 0.06, 0.08, and 0.10 mg / ml.

[0043] 3) Pretreatment of ginseng extract samples Accurately transfer 10.0 ml of ginseng extract into an evaporating dish, evaporate and concentrate to near dryness, then sonicate and extract the ginseng sample with 70% ethanol solution. Transfer the extract to a reaction flask, add a certain volume of concentrated hydrochloric acid to bring the pH to 1, and react in an 80℃ water bath for 3 hours. Transfer to a rotary evaporator, concentrate, and neutralize to pH 7 by adding 6 mol / L NaOH dropwise on a cold water bath. Transfer to a separatory funnel and extract with chloroform four times, 10 ml each time. Combine the extracts, add anhydrous Na2SO4 for dehydration, dry and filter, then evaporate the solvent under reduced pressure. Add 3.0 times the amount of benzoyl chloride derivatizing reagent and 2.0 times the amount of anhydrous pyridine, and incubate in an 80℃ water bath for 2 hours. Remove and dry under reduced pressure with a nitrogen stream. Dissolve in methanol and transfer to a 10 ml volumetric flask, dilute to the mark with methanol, and mix well. Filter the solution through a 0.22 μm microporous membrane, and use the filtrate as the sample solution.

[0044] 4. Optimal chromatographic conditions: By experimenting with various mobile phase compositions and ratios, flow rates, and other factors, the optimal chromatographic conditions were ultimately determined. Chromatographic column: Phenomemex C 18 Analytical column (4.6 × 150 mm, ID 5 μm) Mobile phase: Acetonitrile: Water = 40:60 (V / V) Flow rate: 1.0 mL / min Column temperature: 25℃ Detection wavelength: 230nm Injection volume: 20 μL Subsequently, standard curves for ginsenoside diol and ginsenoside triol were established using a series of derivatized standard solutions at concentrations of 0.01, 0.02, 0.04, 0.06, 0.08, and 0.10 mg / ml.

[0045] The standard curve is: Ginsenoside diol: Ginsenosides: x: concentration (mg / ml) y: peak area (mv·s) 5. Determining the optimal probiotic combination To intuitively screen for beneficial probiotic compositions, we designed groups with different ratios of probiotics and ginseng, selecting three types of Bifidobacterium and three types of Lactobacillus. In six experimental groups with a probiotic ratio of 1:1, ginseng powder was used at both ends, ginseng slices were used in the middle, and other proportions of American ginseng slices and red ginseng slices were also included. The release percentage of ginseng core aglycones was specifically measured under different component conditions to illustrate whether the effect would differ due to different probiotic combinations.

[0046] (1) The relevant designs are as follows: Experimental group consisted of Bifidobacterium longum and Lactobacillus plantarum in a ratio of 1-10:1-6.

[0047] Experimental group 2 consisted of Bifidobacterium bifidum and Lactobacillus acidophilus in a ratio of 1-10:1-6.

[0048] Experimental group 3 consisted of Bifidobacterium animalis and Lactobacillus rhamnosus, with a ratio ranging from 1 to 10: 1 to 6.

[0049] The comparative control group consisted of Bifidobacterium longum and Lactobacillus plantarum in a ratio of 1:4.

[0050] See Tables 3 through 6 for details. Table 3. Bifidobacterium longum and Lactobacillus plantarum groups Table 4. Bifidobacterium and Lactobacillus acidophilus groups Table 5. Animal Bifidobacterium and Lactobacillus rhamnosus groups Table 6. Bifidobacterium longum and Lactobacillus plantarum groups (2) Results In all groups, 10 ml samples were taken every 1 hour to determine the content of ginsenosides and ginsenoside diols in the ginseng extract samples. See Table 7 for details. Table 7. Content of ginsenosides and ginsenoside diol in ginseng extract samples When the ratio of Bifidobacterium to Lactobacillus was between 1~10:1~6 (by weight), and the ratio of ginseng to probiotics (by weight) was above 1:10 (1:1~100), an intestinal dissolution test was conducted for 1 hour, and the final percentage of core aglycone release exceeded 80%.

[0051] Example 2: Determined percentage of probiotic core aglycone release Based on the results of Example 1, the percentage of core aglycone released within 1 hour exceeded 80%. In order to determine the optimal combination again, we designed a complete release experiment to determine the optimal combination.

[0052] Based on the experimental conditions in Example 1, we first prepared three groups each of Bifidobacterium and Lactobacillus (Bifidobacterium longum, Lactobacillus plantarum, Bifidobacterium bifidum: Lactobacillus acidophilus, Bifidobacterium animalis, Lactobacillus rhamnosus) with a ratio of 1:1 and ginseng and probiotics with a ratio of 1:10. A 3-hour intestinal dissolution experiment was simulated, and samples were taken at 20, 40, 60, 120, and 180 minutes to determine the concentration. In the 3-hour intestinal dissolution experiment, groups 6, 16, and 26 showed the best results, as detailed in Table 8.

[0053] Table 8. Percentage of Core Aglycone Release As shown in the table above, when the ratio of Bifidobacterium to Lactobacillus is 1:1 and the ratio of ginseng to probiotics is 1:10, the percentage of core aglycone release exceeds 80%.

[0054] The results of Examples 1 and 2 above show that, with the same proportion of probiotics, the percentage of core aglycone release varies depending on the type and proportion of ginseng added. In summary, the experimental data indicates that, with the same proportion of probiotics, when the ratio of ginseng to probiotics (by weight) is 1:10, the release of core aglycones exceeds 2-3 times. This means that with the addition of a small amount of ginseng, the release of core aglycones achieves the best effect, specifically in groups 6, 16, and 26. While the percentage of core aglycone release reaches 84% ​​with a probiotic ratio of 1:4, the consumption of probiotics also increases.

[0055] The foregoing has shown and described the basic principles, main features, and advantages of the present invention. Those skilled in the art should understand that the present invention is not limited to the above embodiments. The embodiments and descriptions in the specification are merely illustrative of the principles of the invention. Various changes and modifications can be made to the invention without departing from its spirit and scope, and all such changes and modifications fall within the scope of the present invention as claimed. The scope of protection of this invention is defined by the appended claims and their equivalents.

Claims

1. A probiotic composition, characterized in that: The probiotic composition consists of Bifidobacterium and Lactobacillus.

2. The probiotic composition according to claim 1, characterized in that: The ratio of Bifidobacterium to Lactobacillus in the probiotic composition is 1~10:1~6.

3. The probiotic composition according to claim 1, characterized in that: The Bifidobacterium is selected from any one or more of Bifidobacterium adolescentis, Bifidobacterium animalis, lactic acid bacteria, Bifidobacterium, Bifidobacterium bifidum, Bifidobacterium infantis, Bifidobacterium longum, Bifidobacterium animalis, and Bifidobacterium lactis; the Lactobacillus is selected from any one or more of Lactobacillus plantarum, Lactobacillus casei, lactic acid bacteria, Lactobacillus acidophilus, Lactobacillus helveticus, Lactobacillus johnsonii, Lactobacillus lactis, Lactobacillus gasseri, Lactobacillus rhamnosus, and Lactobacillus delbrueckii.

4. A ginsenoside probiotic composition, characterized in that: The ginsenoside probiotic composition comprises ginseng products and a probiotic composition, wherein the probiotic composition contains at least one Bifidobacterium or one Lactobacillus; after the ginsenoside probiotic composition is taken, the probiotics can degrade ginsenosides into ginsenoside core aglycones during human digestion, thereby achieving the effect of improving the bioavailability of ginsenosides.

5. The ginsenoside probiotic composition as described in claim 4, characterized in that: The ratio of the ginseng product and probiotic composition is 1:1 to 100.

6. The ginsenoside probiotic composition as described in claim 4, characterized in that: The conversion rate of the core aglycone in the ginsenoside probiotic composition exceeds 80% within one hour.

7. The use of the probiotic composition as described in claim 1 in any of the following aspects; (1) Preparation of microspheres loaded with ginsenoside probiotic composition; (2) Preparation of nanomedicine delivery systems or devices; (3) Preparation of culture medium for in vitro conversion of ginsenosides; (4) Prepare other health products / dietary supplements.

8. A ginsenoside probiotic preparation with anti-inflammatory and immunomodulatory effects, characterized in that: The formulation comprises the ginsenoside probiotic composition as described in claim 4.

9. The ginsenoside probiotic preparation as described in claim 8, characterized in that: The ginsenoside probiotic composition can be prepared into dosage forms suitable for different populations for treatment.

10. The ginsenoside probiotic preparation as described in claim 8, characterized in that: The dosage form is selected from enteric-coated tablets, enteric-coated capsules, and solid beverages.

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