Ginseng-astragalus membranaceus-containing ginseng-astragalus membranaceus extract essence plant beverage composition and preparation method thereof

Abstract

The invention provides a ginseng-radix astragali extract essence plant beverage composition containing ginseng and radix astragali and a preparation method thereof, and belongs to the technical field of plant beverages. The composition takes an extract obtained by performing similar wild ginseng treatment on artificially planted ginseng as a core, and is matched with compound herbal raw materials such as astragalus membranaceus and angelica sinensis and functional auxiliary materials, the composition is prepared from the following raw materials in parts by weight: 0.8-1.5 parts of similar wild ginseng extract, 3-6 parts of astragalus membranaceus and the like, ginsenoside Rb1 in a finished product is more than or equal to 4.0 mg / g, Rg3 in the finished product is more than or equal to 4.5 mg / g, specific active peptide is contained, astragalus polysaccharide is more than or equal to 1.2 mg / mL, and the content of the active peptide is more than or equal to 1.0 mg / mL. The small molecule active component accounts for not less than 85%, and the solubility is not less than 98%. The core of the preparation method comprises the following steps: carrying out compound strain fermentation on artificially planted ginseng, carrying out compound enzymolysis, carrying out modified ultrafiltration membrane purification similar wild ginseng treatment, carrying out compound herbal synergistic extraction, mixing and blending, and carrying out instantaneous sterilization and filling. The invention solves the problems of low content of effective components, poor absorptivity and insufficient functionality of the traditional ginseng and astragalus drink.

Description

Technical Field

[0001] This invention belongs to the field of plant beverage technology, and more specifically, it relates to a ginseng and astragalus extract plant beverage composition containing ginseng and astragalus and its preparation method. Background Technology

[0002] Ginseng and astragalus-based beverages are widely used in the daily health drink market due to the nourishing effects of ginseng and astragalus. The saponins in ginseng and the polysaccharides in astragalus are the core active substances, possessing functions such as immune regulation, anti-fatigue, and improving physical condition. However, the research and production of ginseng and astragalus-based beverages still face many unresolved issues in current technology.

[0003] Low utilization rate of ginseng's effective components: Traditional processes often use direct crushing and decoction to extract artificially cultivated ginseng. Its saponin components are mostly in their original form, which is difficult to fully convert into easily absorbed active forms. In addition, wild ginseng resources are scarce and expensive, and the efficacy of artificially cultivated ginseng is difficult to reach the level of wild ginseng, resulting in insufficient content of core active ingredients in the product.

[0004] Poor product solubility and absorption rate: Existing beverages contain a lot of macromolecular impurities, and the active ingredients are mostly in the form of macromolecules, which result in low absorption efficiency by the human gastrointestinal tract and are prone to problems such as precipitation and layering, affecting product stability and consumption experience.

[0005] The synergistic effect of compound ingredients is not fully utilized: Most products simply mix extracts of raw materials such as ginseng and astragalus without optimizing the process for dissolving the effective components of the compound ingredients, making it difficult to maximize the synergistic nourishing effects between herbal raw materials.

[0006] The purification process has limited effectiveness: conventional ultrafiltration membranes are prone to fouling and have insufficient retention precision, resulting in a high loss rate of active ingredients in the product. Impurities remain, affecting the purity and functionality of the product. Furthermore, some products rely on artificial pigments and preservatives to enhance their sensory appeal and shelf life, which does not meet the needs of health-conscious consumers.

[0007] Functional lack of data support: Existing similar products mostly focus on sensory flavor, and their core effects such as immune regulation and anti-fatigue lack rigorous animal experiments and testing data verification, resulting in poor efficacy stability and repeatability.

[0008] To address the aforementioned issues, there is an urgent need to develop a ginseng and astragalus-based herbal beverage that utilizes innovative processes to reconstruct the active ingredients of ginseng, enhance the synergistic effects of compound preparations, and optimize product stability and absorption rates, thereby meeting consumers' high-quality demands for healthy and nourishing drinks. Summary of the Invention

[0009] To address the aforementioned technical problems, this invention provides a ginseng and astragalus extract plant beverage composition and its preparation method. This solves the technical problems in traditional ginseng and astragalus beverages, such as low utilization rate of effective components of artificially cultivated ginseng (difficult to achieve the efficacy level of wild ginseng), poor solubility and human absorption rate of active ingredients, insufficient synergistic nourishing effects of compound herbal raw materials, poor product stability due to insufficient purification process, and lack of data support for functionalities such as immune regulation and anti-fatigue.

[0010] The Ginseng and Astragalus Extract Dew herbal beverage composition contains ginseng and astragalus extracts. Its core active ingredient is an extract obtained from artificially cultivated ginseng through a process similar to wild ginseng, combined with compound herbal ingredients and functional excipients. By weight, it comprises:

[0011] Wild ginseng extract 0.8-1.5 parts (prepared from 2-5 parts artificially cultivated ginseng), Astragalus membranaceus 3-6 parts, Angelica sinensis 1-3 parts, Longan 2-4 parts, Lycium barbarum 2-5 parts, Fructooligosaccharides 1-2 parts, Ophiopogon japonicus 1-3 parts, Jujube 2-4 parts, Tangerine peel 0.5-1.5 parts, Polygonatum odoratum 1-2 parts, Lemon powder 0.1-0.3 parts, Purified water 100-150 parts;

[0012] The wild ginseng extract contains ginsenoside Rb1 content ≥ 4.0 mg / g, Rg3 (S-Rg3+R-Rg3) content ≥ 4.5 mg / g, and contains the active peptides shown in SEQ ID NO.1-4;

[0013] The composition contains ≥1.2 mg / mL of Astragalus polysaccharide, ≥85% of small molecule active ingredients (molecular weight ≤3000 Da), and ≥98% solubility.

[0014] Preferably, the raw materials are in the following weight proportions: 1.2 parts of wild ginseng extract (prepared from 3 parts of artificially cultivated ginseng), 4.5 parts of astragalus, 2 parts of angelica, 3 parts of longan, 3.5 parts of wolfberry, 1.5 parts of fructooligosaccharide, 2 parts of ophiopogon japonicus, 3 parts of jujube, 1 part of dried tangerine peel, 1.5 parts of polygonatum odoratum, 0.2 parts of lemon powder, and 120 parts of purified water.

[0015] Preferably, the amino acid sequences of the active peptides shown in SEQ ID NO.1-4 are as follows:

[0016] The four bioactive peptides contained in SEQ ID NO.1 (GenBank:QDI06141.1), SEQ ID NO.2 (GenBank:QDI06141.1), SEQ ID NO.3 (GenBank:AIC33058.1), and SEQ ID NO.4 (GenBank:QDI06141.1) were all present, and the total content of the four bioactive peptides was ≥0.3 mg / g.

[0017] Preferably, the composition has an energy of ≤270kJ / 100mL, a sodium content of ≤70mg / 100mL, is free of artificial colors and preservatives, and has a shelf life of ≥18 months when stored at room temperature in a sealed container.

[0018] Another technical problem to be solved by the present invention is to provide a method for preparing a ginseng and astragalus extract herbal beverage composition, comprising the following key steps:

[0019] S1: Treatment of artificially cultivated ginseng to resemble wild ginseng: S11: The artificially cultivated ginseng was air-dried, ground into powder, and sterilized by steam at 100℃ for 120 minutes, then used as a fermentation substrate; S12: A compound seed liquid was prepared by mixing *Gynostemma pentaphyllum* and *Bifidobacterium adolescentis* at a volume ratio of 2:2-0.3. The seed liquid was prepared using a seed culture medium containing 1.5% ginseng powder, 2.5% peptone, and 2% glucose, and fermented at 37℃ and 60 rpm for 3 days; S13: The compound seed liquid was inoculated at 10% v / v into a fermentation medium containing 12.5% ​​ginseng powder. Culture medium was used for constant temperature fermentation at 35℃ and 80 rpm for 5 days. After sterilization by centrifugation at 8000 rpm for 15 min, the fermentation broth was extracted twice with methanol under hot reflux (2 h each time). The filtrate was concentrated to obtain the first supernatant. S14: 25% BPP protein extraction solution and an equal volume of 25% Tris-saturated phenol reagent were added to the first supernatant. The mixture was centrifuged at 15000 g and 4℃ for 15 min to obtain the upper phenol phase. Five volumes of supersaturated ammonium sulfate-methanol solution (13 mg / mL ammonium sulfate) were added and incubated overnight at -20℃. The precipitate was centrifuged, washed with pre-cooled acetone, and dissolved in RIPA lysis buffer. The second supernatant was obtained; S15: 20 v / v% denaturant (100 mM NH4HCO3 + 8 M urea) and 2 v / v% 1 mg / m LDT were added to the second supernatant, and the mixture was treated at 37℃ for 2 h. Then, 20 m / v% 2-chloroacetamide was added and reacted in the dark for 40 min. Trypsin was then added at a 1:50 (w / w) enzyme ratio and enzymatically hydrolyzed at 37℃ for 24 h. After desalting by modified ultrafiltration membrane and purification by gel chromatography, the mixture was freeze-dried to obtain a wild ginseng-like extract; S2: Synergistic extraction of compound herbal raw materials: Astragalus membranaceus, Angelica sinensis, longan, wolfberry, Ophiopogon japonicus, jujube, Powdered tangerine peel and Solomon's seal rhizome are crushed to 80-100 mesh, mixed according to the formula, and then added to purified water. Soaking is carried out at 25-30℃ for 30-60 minutes, followed by reflux in a water bath at 95-100℃ for 60-90 minutes. The extraction is repeated twice. The extracts are combined and centrifuged at 8000 rpm for 15 minutes to remove impurities. S3: Mixing, blending and refining: The wild ginseng extract, compound extract, fructooligosaccharides and lemon powder are mixed and stirred until completely dissolved. The pH is adjusted to 5.5-6.5. After filtration through a modified ultrafiltration membrane, the mixture is sterilized at 105℃ for 15-20 seconds and aseptically filled to obtain the finished product.

[0020] Preferably, the seed culture medium in S12 further comprises 0.25% disodium hydrogen phosphate, 0.25% potassium dihydrogen phosphate, 0.32% dipotassium hydrogen phosphate, 0.6% sodium acetate, 0.06% magnesium citrate, 0.1% Tween 80, and 0.05% lemon juice; the fermentation culture medium in S13 comprises 3.5% peptone, 4.5% glucose, 0.55% disodium hydrogen phosphate, 0.55% potassium dihydrogen phosphate, 0.15% ammonium citrate, 0.6% sodium acetate, 0.06% magnesium sulfate, 0.023% manganese sulfate, 0.1% Tween 80, 0.25% deoxycholic acid, and 0.2% vitamin K1, with the pH adjusted to 6.0 using citric acid.

[0021] Preferably, the modified ultrafiltration membrane described in S15 is prepared by: placing the ultrafiltration membrane in a 50-100W radio frequency plasma generator and treating it with a mixed gas of α-linolenic acid vapor and nitrogen in a ratio of 1:5-7 (flow rate 12-18 sccm, pressure 15-20 Pa) for 40-50 seconds; then mixing it with an ethanol solution containing 3-4 parts of p-hydroxycinnamic acid, 0.1-0.2 parts of cellulase, and 0.2-0.4 parts of acetic acid, stirring at 65°C for 6-8 hours, washing with anhydrous ethanol, and drying.

[0022] Preferably, in the enzymatic hydrolysis step S15, a complex enzyme is used instead of a single trypsin. The complex enzyme is composed of cellulase, acidic protease, and β-glucanase in a mass ratio of 2:1:1. The amount of enzyme added is 0.5% of the mass of the second supernatant. The enzymatic hydrolysis temperature is 48-52℃ and the enzymatic hydrolysis time is 4.5 days.

[0023] Preferably, during the extraction of compound herbal raw materials in S2, 0.1% vitamin C is added as an antioxidant, and lemon powder (1 / 3 of the formula amount) is added during the second reflux extraction to improve the dissolution rate of active ingredients.

[0024] Compared with the prior art, the present invention has the following beneficial effects:

[0025] Through a process of fermentation with compound microorganisms, compound enzymatic hydrolysis, and purification with modified ultrafiltration membranes, the original saponins of artificially cultivated ginseng are transformed into highly active forms. The finished product contains ginsenosides Rb1 and Rg3 at contents of ≥4.0 mg / g and ≥4.5 mg / g, respectively, and also contains the active peptides shown in SEQ ID NO.1-4 (total content ≥0.3 mg / g) and astragalus polysaccharide at contents of ≥1.2 mg / mL. The content of key active ingredients is 3-6 times higher than that of traditional products, achieving a leap from the efficacy of artificially cultivated ginseng to the level of wild ginseng.

[0026] The wild ginseng-like processing decomposes the active ingredients into small molecules with a molecular weight of ≤3000Da. Combined with the precise retention of the modified ultrafiltration membrane, the finished product has a solubility of ≥98% and a small molecule active ingredient ratio of ≥85%. Compared with the traditional cell wall breaking process, the absorption rate is increased by more than 40%, and the gastrointestinal absorption is faster, avoiding the precipitation problem caused by large molecule impurities.

[0027] Through a compound herbal raw material synergistic extraction process, vitamin C was added as an antioxidant. During the second reflux extraction, some lemon powder was added to promote the dissolution of components such as astragalus polysaccharide, angelica ferulic acid, and wolfberry polysaccharide, forming a synergistic effect with wild ginseng-like extract. Animal experiments have verified that it can significantly increase the thymus index, spleen index, and serum hemolysin level in immunocompromised mice. The weight-bearing swimming time of mice was extended by more than 90% compared with the control group, and the blood lactate concentration was reduced by 40%-52%, demonstrating clear immune regulation and anti-fatigue effects.

[0028] The modified ultrafiltration membrane is modified through radio frequency plasma treatment and graft polymerization, which improves its resistance to fouling and effectively reduces the loss of active ingredients. The finished product has been verified by accelerated testing, and the degradation rate of key components is only 4.5%-5.2% after 3 months. It is estimated that the shelf life is ≥18 months when stored at room temperature in a sealed container. The product does not contain artificial colors or preservatives, has an energy content of ≤270kJ / 100mL, and a sodium content of ≤70mg / 100mL, which meets the standards for healthy beverages and is suitable for a wide range of people (except for certain unsuitable groups).

[0029] This invention integrates multiple core technologies such as compound microbial fermentation, compound enzymatic hydrolysis, and modified ultrafiltration membrane purification. The wild ginseng-like processing technology increases the utilization rate of artificially cultivated ginseng to over 40%, and improves the extraction rate by 30% compared to traditional extraction processes. The process parameters are clear and highly repeatable, and the required equipment is conventional equipment in the food processing field, making it easy to achieve large-scale production. This provides a new path for the high-quality upgrading of ginseng and astragalus beverages. Attached Figure Description

[0030] Figure 1 This is a schematic diagram of the preparation method of the present invention. Detailed Implementation

[0031] The embodiments of the present invention will be described in further detail below with reference to the accompanying drawings and examples. The following examples are for illustrative purposes only and should not be construed as limiting the scope of the invention.

[0032] Please see Figure 1 This invention provides a ginseng and astragalus extract plant beverage composition and its preparation method. To make the technical solution, process parameters and technical effects of this invention clearer and more explicit, the invention will be described in detail below with reference to specific materials, equipment, examples, comparative examples and detection methods.

[0033] Experimental methods not specified in this embodiment were performed in accordance with the Pharmacopoeia of the People's Republic of China (2020 edition) and conventional bioengineering experimental methods; all reagents used were analytical grade or food grade and commercially available.

[0034] Experimental materials and equipment:

[0035] Raw materials and reagents:

[0036]

[0037]

[0038]

[0039] Main equipment;

[0040]

[0041]

[0042] Preparation of key solutions and culture media:

[0043] Seed culture medium: 1.5% (w / v) ginseng powder, 2.5% (w / v) peptone, 2% (w / v) glucose, 0.25% (w / v) disodium hydrogen phosphate (Na2HPO4·12H2O), 0.25% (w / v) potassium dihydrogen phosphate (KH2PO4), 0.32% (w / v) dipotassium hydrogen phosphate (K2HPO4), 0.6% (w / v) sodium acetate (CH3COONa·3H2O), 0.06% (w / v) magnesium citrate (MgC6H6O7·4H2O), 0.1% (v / v) Tween 80, 0.05% (v / v) lemon juice, dissolved in distilled water, sterilized at 121℃ for 20 min, pH at rest (approximately 6.0 after sterilization).

[0044] Fermentation medium: 12.5% ​​(w / v) ginseng powder, 3.5% (w / v) peptone, 4.5% (w / v) glucose, 0.55% (w / v) Na2HPO4·12H2O, 0.55% (w / v) KH2PO4, 0.15% (w / v) ammonium citrate ((NH4)3C6H5O7), 0.6% (w / v) CH3COONa·3H2O, 0.06% (w / v) magnesium sulfate (MgSO4·7H2O), 0.023% (w / v) manganese sulfate (MnSO4·H2O), 0.1% (w / v) Tween 80, 0.25% (w / v) deoxycholic acid, 0.2% (w / v) vitamin K1. Adjust the pH to 6.0 with citric acid. Sterilize at 121℃ for 20 min.

[0045] Denaturant: 100mMNH4HCO3 + 8M urea, prepared with distilled water, and sterilized by filtration through a 0.22μm filter membrane.

[0046] DTT solution: 1 mg / mL, prepared with the above denaturant, and used immediately after preparation.

[0047] 2-Chloroacetamide solution: 20% (m / v), prepared with distilled water, sterilized by filtration through a 0.22 μm filter membrane.

[0048] Example 1: Basic Formulation and Standard Process

[0049] Raw material ratio (by weight, total formula volume 10L):

[0050]

[0051]

[0052] Preparation steps;

[0053] S1: Artificially cultivated ginseng undergoes a process similar to wild ginseng;

[0054] S11 Ginseng Pretreatment: 300g of artificially cultivated ginseng is air-dried (moisture content ≤10%), pulverized to 80 mesh using an ultra-micro pulverizer (25Hz, 80s), steam-sterilized at 100℃ for 120min, and vacuum-dried (60℃, -0.08MPa) until moisture content ≤5% for later use.

[0055] Preparation of S12 composite seed liquid:

[0056] Ginsenoside Rhizome and Bifidobacterium adolescentis were inoculated into seed culture medium and anaerobic fermented at 37°C and 60 rpm for 3 days to obtain single-strain seed culture (strain concentration ≥10). 8 (CFU / mL);

[0057] Mix the two bacterial seed solutions (200 mL of *Gynostemma pentaphyllum* and 30 mL of *Bifidobacterium adolescentis*) at a volume ratio of 2:0.3 to obtain a compound seed solution (total bacterial concentration 1.05 × 10⁻⁶). 8 (CFU / mL).

[0058] S13 Fermentation and Preliminary Extraction:

[0059] Add 12.5% ​​(w / v) ginseng powder (i.e., 300g pretreated ginseng powder + 2100mL distilled water) to the fermentation tank, inoculate with 10% (v / v) compound seed liquid (240mL), and ferment at a constant temperature of 35℃ and 80rpm for 5 days.

[0060] The fermentation broth was centrifuged at 8000 rpm for 15 min (4℃) to remove the bacterial cells (the wet weight of the precipitate was about 18 g, which was discarded), and 2200 mL of supernatant was obtained.

[0061] The supernatant was extracted twice by hot reflux with methanol (solid-to-liquid ratio 1:3, v / v) (2 h each time, 80 °C), and filtered while hot (0.45 μm filter membrane). The filtrates were combined into 6200 mL and concentrated under reduced pressure (60 °C, -0.09 MPa) to 500 mL to obtain the first supernatant (density 1.12 g / mL).

[0062] S14 protein extraction and ammonium sulfate precipitation:

[0063] Add 25% (v / v) BPP protein extraction solution (125 mL) and an equal volume of 25% (v / v) Tris-saturated phenol reagent (625 mL) to the first supernatant (500 mL), shake to mix for 10 min, centrifuge at 15000 g and 4 °C for 15 min to obtain the upper phenol phase (about 580 mL).

[0064] Add 5 times the volume of supersaturated ammonium sulfate-methanol solution (ammonium sulfate concentration 13 mg / mL, i.e. 580 mL × 5 = 2900 mL) to the phenol phase and let it stand overnight (12 h) at -20 °C.

[0065] Centrifuge at 15000g and 4℃ for 5min to obtain a precipitate (wet weight about 28g). Wash three times with pre-cooled acetone (-20℃, 50mL×3), discarding the supernatant after each centrifugation.

[0066] Add 100 mL of RIPALysis Buffer (containing 1% protease inhibitor) to the precipitate, vortex to mix, let stand at room temperature for 2 h, centrifuge at 20000 g at room temperature for 30 min, and obtain 85 mL of supernatant, which is the second supernatant (protein concentration was 4.2 mg / mL as determined by BCA method).

[0067] S15 enzymatic digestion and purification:

[0068] Add 20% (v / v) denaturant (17 mL) and 2% (v / v) 1 mg / mL LDTT (1.7 mL) to the second supernatant (85 mL), and treat in a water bath at 37°C for 2 h;

[0069] Add 20% (m / v) 2-chloroacetamide (1.7 g, dissolved in 8.5 mL distilled water) and react at room temperature in the dark for 40 min;

[0070] Add 7× buffer (100mMNH4HCO3, 602mL) to dilute the denaturant, add trypsin (0.0714g, 1mg / mL solution, 71.4mL) at an enzyme ratio of 1:50 (w / w), and incubate at 37℃ for 24h to obtain 780mL of enzymatic hydrolysate.

[0071] Preparation of modified ultrafiltration membranes:

[0072] A 3000Da PVDF ultrafiltration membrane was placed in a radio frequency plasma generator and a mixed gas of α-linolenic acid vapor and nitrogen gas = 1:6 (v / v) was introduced (flow rate 15 sccm, power 70W, gas pressure 18Pa) and treated for 45 seconds to obtain an ultrafiltration membrane with double bond groups.

[0073] Add 3.5g of p-hydroxycinnamic acid, 0.15g of cellulase, 0.3g of acetic acid, and 280g of ethanol to a stirred tank and stir at 65℃ for 3 hours to obtain a mixed solution;

[0074] Add 25g of ultrafiltration membrane with double bond groups and 0.03g of potassium peroxide, stir at 65℃ for 7h, wash 3 times with anhydrous ethanol (50mL each time), and dry at 60℃ for 2h to obtain the modified ultrafiltration membrane.

[0075] The enzymatic hydrolysate was filtered through a modified ultrafiltration membrane (operating pressure 0.15 MPa, temperature 30℃), and 750 mL of permeate was collected. The permeate was concentrated to 50 mL under reduced pressure and then freeze-dried (-50℃, -0.1 MPa, 24 h) to obtain 122 g of wild ginseng extract (yield 40.7%, meeting the target yield of 40%).

[0076] S2 compound herbal raw material synergistic extraction:

[0077] Mix Astragalus membranaceus (450g), Angelica sinensis (200g), longan (300g), wolfberry (350g), Ophiopogon japonicus (200g), jujube (300g), dried tangerine peel (100g), and Polygonatum odoratum (150g), add 12000mL of purified water, and soak at 28℃ for 45 minutes.

[0078] Extract by reflux in a water bath at 98℃ for 75 min, and filter (using a 0.45μm filter membrane) to obtain 9500 mL of the first extract;

[0079] Add 8000 mL of purified water to the residue, reflux at 98 °C for 60 min, and filter to obtain 7200 mL of the second extract;

[0080] Combine the two extracts (16700 mL), centrifuge at 8000 rpm for 15 min (4℃), remove the residue (wet weight about 280 g, discard), and obtain 16200 mL of compound extract (density 1.03 g / mL, the concentration of Astragalus polysaccharide was 1.8 mg / mL as determined by the phenol-sulfuric acid method).

[0081] S3 blending and refining:

[0082] Add the wild ginseng extract (122g), compound extract (16200mL), fructooligosaccharides (150g), and lemon powder (20g) to a mixing jar and stir at 30℃ for 30 minutes (50rpm) until completely dissolved.

[0083] Adjust the pH to 6.0 using a 10% citric acid solution;

[0084] The prepared solution was filtered through a modified ultrafiltration membrane (0.12 MPa, 30℃) to obtain 16000 mL of clear filtrate;

[0085] The filtrate was sterilized by a flash sterilizer (105℃, 18s) and aseptically filled (50mL / bottle, 320 bottles) to obtain the finished product, Ginseng and Astragalus Extract.

[0086] Example 2: Optimized Formula (Increased Lactobacillus acidophilus)

[0087] The only difference from Example 1 is:

[0088] S12 compound seed liquid: Ginseng Kernella: Bifidobacterium adolescentis: Lactobacillus acidophilus = 2:2:0.3 (volume ratio), the bacterial concentration after mixing is 1.12×10. 8 CFU / mL;

[0089] The proportions of other raw materials and the preparation steps were the same as in Example 1. Finally, 125g of wild ginseng extract (yield 41.7%) was obtained, and 320 bottles of finished product were produced.

[0090] Example 3: Process Optimization (Compound Enzyme Replaces Trypsin)

[0091] The only difference from Example 1 is:

[0092] S15 enzymatic hydrolysis steps: Replace trypsin with a compound enzyme (cellulase: acidic protease: β-glucanase = 2:1:1, w / w), the amount of enzyme added is 0.5% of the mass of the second supernatant (0.85mL × 1.03g / mL × 0.5% ≈ 0.0043g, i.e. 4.3mg), the enzymatic hydrolysis temperature is 50℃, and the enzymatic hydrolysis time is 4.5 days;

[0093] The proportions of other raw materials and the preparation steps were the same as in Example 1. Finally, 128g of wild ginseng extract (yield 42.7%) was obtained, and 320 bottles of finished product were produced.

[0094] Comparative Example 1: Treatment of wild ginseng without classification (extracted from ordinary ginseng)

[0095] Difference: The wild ginseng-like treatment in S1 is omitted. 300g of artificially cultivated ginseng is directly crushed and then extracted with 12000mL of purified water and compound raw materials (steps are the same as in S2).

[0096] The proportions of other raw materials and the preparation steps (such as using ordinary PVDF membrane for ultrafiltration without modification) are the same as in Example 1, yielding 320 bottles of finished product.

[0097] Comparative Example 2: Unmodified ultrafiltration membrane (using ordinary ultrafiltration membrane)

[0098] Difference: Both S15 and S3 use unmodified 3000Da PVDF ultrafiltration membranes (without radio frequency plasma treatment + p-hydroxycinnamic acid grafting);

[0099] The proportions of other raw materials and the preparation steps were the same as in Example 1, yielding 98g of wild ginseng extract (yield 32.7%) and 320 bottles of finished product.

[0100] Comparative Example 3: Single-strain fermentation (without Ginseng Kernella)

[0101] Difference: S12 uses only a single-strain seed liquid of Bifidobacterium adolescentis (without artificially cultivated Gynostemma pentaphyllum), and the fermentation time is 5 days;

[0102] The proportions of other raw materials and the preparation steps were the same as in Example 1, yielding 85g of wild ginseng extract (yield 28.3%) and 320 bottles of finished product.

[0103] Comparative Example 4: Commercially available similar products (Ginseng and Astragalus Oral Liquid)

[0104] We selected commercially available ginseng and astragalus oral liquid (10mL / vial, ingredients: ginseng, astragalus, sucrose, and purified water) as a market control sample.

[0105] Performance testing methods and formulas:

[0106] Key component content testing:

[0107] Detection of ginsenosides (Rb1, Rg3) (HPLC method):

[0108] Chromatographic conditions: Waters ACQUITYUPLCBEHC18 column (100 mm × 2.1 mm, 1.7 μm);

[0109] Mobile phases A (0.01% aqueous acetic acid solution) and B (0.01% acetic acid-acetonitrile = 20:80);

[0110] Flow rate: 0.5 mL / min;

[0111] Column temperature 40℃;

[0112] Injection volume: 20 μL;

[0113] Elution program: 0-10 min 90% A, 10-50 min 90% A → 0% A, 50-65 min 0% A, 65-70 min 0% A → 90% A;

[0114] The detection wavelength is 203nm.

[0115] Standard curve: Prepare standard solutions of Rb1 (0.1, 0.2, 0.5, 1.0, 2.0 mg / mL) and Rg3 (S-Rg3+R-Rg3, 0.05, 0.1, 0.2, 0.5, 1.0 mg / mL) respectively, and plot the peak area (Y) vs. concentration (X, mg / mL) standard curve to obtain the regression equation:

[0116] Rb1: Y = 12543X + 128.6(R 2 =0.9998)

[0117] Rg3: Y = 11876X + 95.3(R 2 =0.9997)

[0118] Sample testing: Take 1 mL of the finished product, dilute it 10 times with methanol, filter it through a 0.22 μm filter membrane, inject the sample for testing, and substitute it into the regression equation to calculate the content.

[0119] Detection of bioactive peptide content (UPLC-MS / MS method):

[0120] Chromatographic conditions:

[0121] Agi lentEcl ipseXDB-C18 column (150mm×4.6mm, 2.2μm);

[0122] Mobile phases A (0.1% formic acid aqueous solution) and B (0.1% formic acid-acetonitrile = 20:80);

[0123] Flow rate: 0.35 mL / min;

[0124] Column temperature 60℃;

[0125] Injection volume: 5 μL;

[0126] Elution program: 0-60 min 99% A → 66% A, 60-75 min 66% A → 53% A, 75-90 min 10% A.

[0127] Mass spectrometry conditions: Orbitrap mass analyzer, scan range 300-1500 m / z, HCD collision energy 30%, AGC = 4 × 10⁻⁶ m / z. 5 .

[0128] Quantitative method: A standard curve was plotted using the standard peptide of SEQ ID NO.1-4 (purity ≥98%), and the total content of the four active peptides in the sample was calculated.

[0129] Astragalus polysaccharide detection (phenol-sulfuric acid method):

[0130] Standard curve: Prepare glucose standard solutions (0.02, 0.04, 0.06, 0.08, 0.10 mg / mL), add 1 mL of 5% phenol solution and 5 mL of concentrated sulfuric acid, react at 40℃ for 20 min, and measure absorbance (A) at 490 nm. The regression equation is: Y = 5.23X + 0.012(R²). 2 =0.9996).

[0131] Sample testing: Take 2 mL of the finished product, add 5 mL of distilled water, extract in an 80℃ water bath for 30 min, centrifuge and take the supernatant, add anhydrous ethanol to precipitate the polysaccharide, dissolve and measure the absorbance according to the standard curve method, and calculate the content of Astragalus polysaccharide.

[0132] Physicochemical performance testing:

[0133] Solubility:

[0134] Formula: Solubility (%) = (Mass of dissolved solids / Total mass of solids in the sample) × 100%

[0135] Procedure: Take 100 mL of the finished product, centrifuge at 6000 rpm for 10 min, collect the supernatant, dry under reduced pressure (80℃, -0.09 MPa) to constant weight, and weigh the dissolved solid mass (m1); take another 100 mL of the finished product and dry it directly to constant weight, weigh the total solid mass (m0), and substitute it into the formula for calculation.

[0136] Percentage of small molecule active ingredients (GPC method):

[0137] Chromatographic conditions: Agi Len Plaquagel-OHMIXED column (300 mm × 7.5 mm); mobile phase 0.1 M NaNO3 aqueous solution; flow rate 0.8 mL / min; column temperature 40 °C; differential refractive index detector.

[0138] Formula: Percentage of small molecules (%) = (Sum of peak areas of molecules with molecular weight ≤ 3000 Da / Total peak area) × 100%

[0139] Procedure: Take 5 mL of the finished product, concentrate it to 1 mL, inject it for detection, and analyze the peak area using a GPC workstation.

[0140] Stability (Shelf life prediction):

[0141] Accelerated testing: The finished product was stored at 40℃ and 75% RH for 3 months, and the content of key components (Rb1, Rg3, and Astragalus polysaccharide) was tested monthly to calculate the degradation rate.

[0142] Degradation rate (%) = (Initial content - Content after storage) / Initial content × 100%

[0143] Shelf life calculation: based on Q 10Based on the rule (the reaction rate doubles for every 10°C increase in temperature), calculate the shelf life at room temperature (25°C):

[0144] Formula: t 25 =t 40 ×Q 10 ^((40-25) / 10)(Q 10 Take 2.0, for regular food Q 10 value)

[0145] Functional testing (animal experiments):

[0146] Immune regulation function (refer to the "Technical Specifications for Inspection and Evaluation of Health Foods"):

[0147] Animal model: KM male mice (18-22g) were intraperitoneally injected with cyclophosphamide (40mg / kg) to establish an immunodeficiency model. They were divided into a blank group (normal mice), a model group (immune deficiency, no drug administration), an example group (immune deficiency, gavage administration of finished product 10mL / kg / d), and a control group (same as the example group). Each group consisted of 10 mice, and the mice were administered the drug by gavage for 28 consecutive days.

[0148] Detection indicators: thymus index (thymus mass / body mass, mg / g), spleen index (spleen mass / body mass, mg / g), serum hemolysin level (U / mL, half-hemolysis method).

[0149] Anti-fatigue function (weighted swimming test):

[0150] Animal model: ICR male mice (20-22g) were divided into the example group (10mL / kg / d of finished product by gavage), the comparative group, and the blank group (10 mice by gavage). Each group was administered the product by gavage for 14 consecutive days.

[0151] Detection indicators: weighted swimming time (mice were loaded with 5% of their body weight, and the time from swimming to sinking was recorded), and blood lactate concentration (blood was collected 30 minutes after swimming, and lactate was detected using a lactate kit).

[0152] Test results and analysis:

[0153] Key component content results:

[0154]

[0155]

[0156] Analysis: The contents of key components in Examples 1-3 were significantly higher than those in the comparative examples (p<0.05). Among them, Example 3 (compound enzyme) had the highest contents of Rb1, Rg3, and active peptides due to improved enzymatic hydrolysis efficiency. Comparative Example 1 (without wild ginseng-like treatment) had almost no active peptides due to the lack of fermentation and enzymatic hydrolysis, and the saponin content was only 1 / 3-1 / 6 of that in Example 1, proving that the wild ginseng-like treatment is the core innovation. Comparative Example 2 (ordinary ultrafiltration membrane) had a low membrane retention efficiency, resulting in a loss of about 30% of active ingredients, demonstrating the advantages of the modified ultrafiltration membrane.

[0157] Physicochemical performance results:

[0158]

[0159]

[0160] Analysis: The solubility of Examples 1-3 is ≥98%, and the proportion of small molecules is ≥86%, which is significantly higher than that of the comparative example (p<0.05). Moreover, the accelerated degradation rate is low, and the estimated shelf life is ≥18 months, which meets the requirements of Claim 1. Comparative Example 1 has more macromolecular impurities, lower solubility and proportion of small molecules, and poor stability because it does not have wild ginseng-like treatment. This proves that the process of the present invention can improve the physicochemical properties of the product.

[0161] Functional test results:

[0162] (1) Results of immune regulation function:

[0163]

[0164]

[0165] (2) Results of anti-fatigue function

[0166]

[0167]

[0168] Analysis: The thymus index, spleen index, and serum hemolysin levels of Examples 1-3 were close to those of the blank group and significantly higher than those of Comparative Examples 1 and 4 (p<0.05), proving that they can restore the immune function of immunocompromised mice. In the anti-fatigue test, the weighted swimming time of the Example group was 1.9-2.2 times that of the blank group, and the blood lactate concentration was reduced by 40%-52%, which was significantly better than that of the Comparative Example, demonstrating the immunomodulatory and anti-fatigue functions of the present invention and meeting the application requirements.

[0169] This invention utilizes an innovative process involving wild ginseng-like processing (compound microbial fermentation + enzymatic hydrolysis), modified ultrafiltration membrane purification, and synergistic extraction of compound herbs to prepare a ginseng and astragalus extract plant beverage composition. This composition exhibits high content of key components (ginsenosides, active peptides, and astragalus polysaccharides), excellent solubility and small molecule proportions, good stability, and significant immunomodulatory and anti-fatigue functions. Comparison of examples and comparative examples demonstrates that the core processes of this invention (wild ginseng-like processing and modified ultrafiltration membrane) are crucial to achieving the product's superiority, demonstrating outstanding inventiveness and practicality. #Ginseng and Astragalus Extract Plant Beverage Composition and its Preparation Method.

[0170] The embodiments of the present invention are given for illustrative and descriptive purposes only, and are not intended to be exhaustive or to limit the invention to the forms disclosed. Many modifications and variations will be apparent to those skilled in the art. The embodiments were chosen and described in order to better illustrate the principles and practical application of the invention, and to enable those skilled in the art to understand the invention and to design various embodiments with various modifications suitable for a particular purpose.

Claims

1. A ginseng and astragalus extract herbal beverage composition, characterized in that, The composition uses an extract obtained from artificially cultivated ginseng through a process similar to wild ginseng as its core active ingredient, combined with compound herbal raw materials and functional excipients, and comprises, by weight: Wild ginseng extract 0.8-1.5 parts, Astragalus membranaceus 3-6 parts, Angelica sinensis 1-3 parts, Longan 2-4 parts, Lycium barbarum 2-5 parts, Fructooligosaccharides 1-2 parts, Ophiopogon japonicus 1-3 parts, Jujube 2-4 parts, Tangerine peel 0.5-1.5 parts, Polygonatum odoratum 1-2 parts, Lemon powder 0.1-0.3 parts, Purified water 100-150 parts; The wild ginseng extract contains ginsenoside Rb1 content ≥ 4.0 mg / g, Rg3 (S-Rg3+R-Rg3) content ≥ 4.5 mg / g, and contains the active peptides shown in SEQ ID NO.1-4; The composition contains ≥1.2 mg / mL of Astragalus polysaccharide, ≥85% of small molecule active ingredients, and has a solubility of ≥98%.

2. A method for preparing a ginseng and astragalus extract herbal beverage composition, characterized in that, This includes the following key steps: S1: Treatment of artificially cultivated ginseng to resemble wild ginseng: S11: Artificially cultivated ginseng is air-dried, ground into powder, and then sterilized by steam at 100℃ for 120 minutes, which is then used as a fermentation substrate. S12: A compound seed liquid was prepared by mixing Gynostemma pentaphyllum and Bifidobacterium adolescentis at a volume ratio of 2:2-0.

3. The seed liquid was prepared by fermenting a seed culture medium containing 1.5% ginseng powder, 2.5% peptone and 2% glucose at 37℃ and 60rpm for 3 days. S13: The compound seed liquid was inoculated into a fermentation medium containing 12.5% ​​ginseng powder at 10% v / v and fermented at 35℃ and 80 rpm for 5 days. After the fermentation liquid was centrifuged at 8000 rpm for 15 min to remove bacteria, it was extracted twice with methanol under hot reflux. The filtrate was concentrated to obtain the first supernatant. S14: Add 25% BPP protein extraction solution and an equal volume of 25% Tris-saturated phenol reagent to the first supernatant, centrifuge at 15000g and 4℃ for 15min to obtain the upper phenol phase, add 5 times the volume of supersaturated ammonium sulfate-methanol solution and incubate at -20℃ overnight, centrifuge the precipitate, wash with pre-cooled acetone and dissolve in RIPA lysis buffer to obtain the second supernatant. S15: Add 20 v / v% denaturant and 2 v / v% 1 mg / mL LDT to the second supernatant, treat at 37°C for 2 h, then add 20 mL / v% 2-chloroacetamide and react in the dark for 40 min. Then add trypsin at a 1:50 (w / w) enzyme ratio and enzymatically hydrolyze at 37°C for 24 h. After desalting by modified ultrafiltration membrane, purifying by gel chromatography, and freeze-drying, obtain wild ginseng-like extract. S2: Synergistic extraction of compound herbal raw materials: Astragalus membranaceus, Angelica sinensis, longan, wolfberry, Ophiopogon japonicus, jujube, tangerine peel, and Polygonatum odoratum are pulverized to 80-100 mesh, mixed according to the formula, and then purified water is added. The mixture is soaked at 25-30℃ for 30-60 min, and then refluxed in a water bath at 95-100℃ for 60-90 min. The extraction is repeated twice. The extracts are combined and centrifuged at 8000 rpm for 15 min to remove impurities. S3: Mixing, blending and refining: Mix wild ginseng extract, compound extract with fructooligosaccharides and lemon powder, stir until completely dissolved, adjust pH to 5.5-6.5, filter through modified ultrafiltration membrane, sterilize at 105℃ for 15-20 seconds, and aseptically fill to obtain the finished product.

3. The ginseng and astragalus extract herbal beverage composition according to claim 1, characterized in that, The specific weight parts of the raw materials are as follows: 1.2 parts of wild ginseng extract, 4.5 parts of astragalus, 2 parts of angelica, 3 parts of longan, 3.5 parts of wolfberry, 1.5 parts of fructooligosaccharides, 2 parts of ophiopogon japonicus, 3 parts of jujube, 1 part of dried tangerine peel, 1.5 parts of Solomon's seal, 0.2 parts of lemon powder, and 120 parts of purified water.

4. The ginseng and astragalus extract herbal beverage composition according to claim 1, characterized in that, The amino acid sequences of the active peptides shown in SEQ ID NO.1-4 are SEQ ID NO.1, SEQ ID NO.2, SEQ ID NO.3, and SEQ ID NO.4, respectively, and the total content of the four active peptides is ≥0.3mg / g.

5. The preparation method of the ginseng and astragalus extract herbal beverage composition according to claim 2, characterized in that, The seed culture medium described in S12 also contains 0.25% disodium hydrogen phosphate, 0.25% potassium dihydrogen phosphate, 0.32% dipotassium hydrogen phosphate, 0.6% sodium acetate, 0.06% magnesium citrate, 0.1% Tween 80, and 0.05% lemon juice; The fermentation medium described in S13 contains 3.5% peptone, 4.5% glucose, 0.55% disodium hydrogen phosphate, 0.55% potassium dihydrogen phosphate, 0.15% ammonium citrate, 0.6% sodium acetate, 0.06% magnesium sulfate, 0.023% manganese sulfate, 0.1% Tween 80, 0.25% deoxycholic acid, and 0.2% vitamin K1, with the pH adjusted to 6.0 using citric acid.

6. The preparation method of the ginseng and astragalus extract plant beverage composition according to claim 2, characterized in that, The method for preparing the modified ultrafiltration membrane described in S15 is as follows: The ultrafiltration membrane was placed in a 50-100W radio frequency plasma generator to generate α-linolenic acid vapor. Treat with a nitrogen-to-gas mixture of 1:5-7 for 40-50 seconds; Then mix with an ethanol solution containing 3-4 parts of p-hydroxycinnamic acid, 0.1-0.2 parts of cellulase, and 0.2-0.4 parts of acetic acid, stir at 65°C for 6-8 hours, wash with anhydrous ethanol and dry.

7. The preparation method of the ginseng and astragalus extract herbal beverage composition according to claim 2, characterized in that, In the enzymatic hydrolysis step of S15, a complex enzyme is used instead of a single trypsin. The complex enzyme is composed of cellulase, acidic protease and β-glucanase in a mass ratio of 2:1:

1. The amount of enzyme added is 0.5% of the mass of the second supernatant. The enzymatic hydrolysis temperature is 48-52℃ and the enzymatic hydrolysis time is 4.5 days.

8. The ginseng and astragalus extract herbal beverage composition according to claim 1, characterized in that, The composition has an energy of ≤270kJ / 100mL, a sodium content of ≤70mg / 100mL, contains no artificial colors or preservatives, and has a shelf life of ≥18 months when stored at room temperature in a sealed container.

9. The preparation method of the ginseng and astragalus extract herbal beverage composition according to claim 2, characterized in that, In the extraction of compound herbal raw materials in S2, 0.1% vitamin C is added as an antioxidant, and lemon powder (1 / 3 of the formula amount) is added during the second reflux extraction to improve the dissolution rate of active ingredients.

10. The application of the ginseng and astragalus extract herbal beverage composition according to claim 1, characterized in that, The composition is used to prepare a plant-based beverage with immune-regulating, anti-fatigue, and complexion-improving effects, with a daily consumption not exceeding 200 mL.

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