A Brazilian ginseng-guarana encapsulated water extract, its preparation method and application

Abstract

This invention provides a Brazilian ginseng-guarana encapsulated aqueous extract, its preparation method, and its application, belonging to the field of anti-fatigue product development technology. The preparation method of the Brazilian ginseng-guarana encapsulated aqueous extract includes: mixing Brazilian ginseng and guarana at a mass ratio of 1:9, adding water for ultrasonic-enzyme pretreatment, extracting and concentrating after pretreatment to obtain the Brazilian ginseng-guarana encapsulated aqueous extract; encapsulating the Brazilian ginseng-guarana encapsulated aqueous extract, and freeze-drying to obtain the Brazilian ginseng-guarana encapsulated aqueous extract. This invention combines ultrasonic-enzyme synergistic pretreatment with optimized encapsulation technology to simultaneously improve the dissolution rate, stability, and bioavailability of the active ingredients of Brazilian ginseng and guarana. The process is simple, cost-controllable, and suitable for large-scale production.

Description

Technical Field

[0001] This invention belongs to the field of anti-fatigue product development technology, and in particular relates to a Brazilian ginseng-guarana encapsulated composite water extract, its preparation method and application. Background Technology

[0002] Brazilian ginseng, a traditional tonic ingredient, contains components such as ecdysterone, which have excellent tonic and conditioning effects; guarana, rich in caffeine, is a common ingredient for stimulating energy and combating fatigue. The combination of the two can achieve a synergistic effect of nourishing and combating fatigue.

[0003] Existing technologies include preparation methods for guarana complex water extracts involving Brazilians, but these methods still have significant technical drawbacks: First, the active ingredients are not fully dissolved. Traditional water extraction or single pretreatment (ultrasound or enzymatic hydrolysis only) cannot completely break down plant cell walls, resulting in low dissolution rates of core components such as caffeine and ecdysterone. Second, the storage stability is poor. Impurities such as macromolecular polysaccharides (cellulose, pectin) and oxidases remaining in the water extract can promote the polymerization and oxidative degradation of active ingredients. After 6 months of storage at room temperature, the retention rate of active ingredients is only about 70%. Summary of the Invention

[0004] In view of this, the purpose of this invention is to provide a Brazilian ginseng-guarana encapsulated composite water extract and its preparation method. By combining ultrasonic-composite enzyme synergistic pretreatment with optimized encapsulation technology, the dissolution rate, stability and bioavailability of the active ingredients of Brazilian ginseng and guarana are simultaneously improved. Moreover, the process is simple, the cost is controllable, and it is suitable for large-scale production.

[0005] To achieve the above-mentioned objectives, the present invention provides the following technical solution: A method for preparing a Brazilian ginseng-guarana encapsulated aqueous extract includes the following steps: Brazilian ginseng and guarana were mixed at a mass ratio of 1:9, and water was added for ultrasonic-complex enzyme pretreatment. After pretreatment, the mixture was extracted and concentrated to obtain a Brazilian ginseng-guarana complex water extract. The ultrasonic-complex enzyme pretreatment included ultrasonic treatment at 200-300 W and 35-45 kHz for 5-15 min, followed by the addition of 0.08%-0.12% of cellulase-xylanase at a mass ratio of (6:4)-(8:2), enzymatic hydrolysis at 40-50 ℃ for 20-40 min, and inactivation. Brazilian ginseng-guarana complex water extract was encapsulated and freeze-dried to obtain Brazilian ginseng-guarana encapsulated complex water extract; the encapsulation wall material was a mixture of maltodextrin and β-cyclodextrin, and the mass ratio of maltodextrin to β-cyclodextrin was (7:3)-(9:1).

[0006] Preferably, the Brazilian ginseng slices are 1-3 mm thick, and the guarana is pulverized to a particle size of 1-5 mm after the brown outer seed coat is removed.

[0007] Preferably, the material-to-liquid ratio of the ultrasound-composite enzyme pretreatment is 1 g: 30-50 mL.

[0008] Preferably, during the extraction, water is added to 100-133 times the total mass of the mixed raw materials, the extraction time is 30-90 minutes, and the extraction is performed 1-3 times.

[0009] Preferably, the extraction is followed by filtration, and the mixed filtrate is concentrated to 5%-25% of its original volume.

[0010] Preferably, the encapsulated wall material aqueous solution has a mass fraction of 10%-15%, and the volume ratio of the Brazilian ginseng-guarana composite water extract to the wall material aqueous solution is 1:2-1:4.

[0011] Preferably, the freeze-drying includes: pre-freezing the encapsulated mixture to -40 to -60 °C for 4 to 6 h, followed by freeze-drying at a vacuum of 10 to 30 Pa and a drying temperature of -20 to -10 °C for 24 to 36 h, resulting in a powder with a particle size of 20 to 80 μm and an encapsulation rate of ≥85%.

[0012] This invention also provides the Brazilian ginseng-guarana encapsulated water extract prepared by the above method and its application in the preparation of anti-fatigue functional beverages.

[0013] The present invention also provides an anti-fatigue functional beverage, comprising the following components by mass concentration: 25%-35% of the above-mentioned Brazilian ginseng-guarana encapsulated complex water extract, 3%-4% maltitol, 0.01%-0.02% citric acid, and the balance being water.

[0014] Compared with the prior art, the present invention has the following beneficial effects: This invention utilizes the cavitation effect of ultrasound and the degradation effect of compound enzymes to achieve a synergistic effect, increasing the dissolution rate of caffeine and ecdysterone by more than 78% compared to traditional water extraction or single pretreatment. At the same time, it degrades macromolecular polysaccharide impurities, deactivates oxidases, and reduces the factors that induce the polymerization and oxidation of active ingredients, achieving a retention rate of more than 95% after 6 months of storage at room temperature.

[0015] This invention uses a composite wall material of maltodextrin and β-cyclodextrin, which solves the problems of low encapsulation rate or poor solubility of single wall materials. The encapsulation rate is ≥85%, and the solubility in water is ≥90%. It provides a physical barrier for active ingredients, ensures the dispersibility of subsequent beverage formulations, and achieves intestinal controlled release, thereby improving bioavailability.

[0016] The in vitro intestinal release rate of the encapsulated composite water extract prepared by this invention is ≥80%. Animal experiments have verified that the high-dose group of mice had a 24.3% longer swimming time under load compared with the unmodified beverage, a 8.7% lower serum urea content, and a 9.3% smaller area under the blood lactate curve, showing better anti-fatigue effects and no adverse effects on animal growth and development, with good safety. Detailed Implementation

[0017] This invention provides a method for preparing a Brazilian ginseng-guarana encapsulated composite water extract, comprising the following steps: Brazilian ginseng and guarana were mixed at a mass ratio of 1:9, and water was added for ultrasonic-complex enzyme pretreatment. After pretreatment, the mixture was extracted and concentrated to obtain a Brazilian ginseng-guarana complex water extract. The ultrasonic-complex enzyme pretreatment included ultrasonic treatment at 200-300 W and 35-45 kHz for 5-15 min, followed by the addition of 0.08%-0.12% of cellulase-xylanase at a mass ratio of (6:4)-(8:2), enzymatic hydrolysis at 40-50 ℃ for 20-40 min, and inactivation.

[0018] The cell walls of Brazilian ginseng and guarana contain a dense lignocellulose-hemicellulose cross-linked network structure. This structure is the core physical barrier preventing the dissolution of ecdysterone (Brazilian ginseng) and caffeine (guarana). Cellulose forms the backbone of the cell wall, while hemicellulose, mainly composed of xylan, is cross-linked with the cellulose backbone through glycosidic bonds, forming a three-dimensional network structure that tightly encapsulates the active ingredients within the cell wall. Single cellulases can only degrade the cellulose backbone and cannot destroy the cross-linking sites between xylan and cellulose, leaving the cell wall partially dense and hindering the full release of active ingredients. In contrast, xylanases can specifically degrade the xylan side chains in hemicellulose, working synergistically with cellulases to achieve targeted disruption of the cross-linked structure.

[0019] The 7:3 cellulase-xylanase mass ratio selected in this invention is based on the optimized proportion of cell wall components in the two raw materials. At this ratio, cellulase can efficiently degrade the main chain backbone, while xylanase precisely targets the cross-linked side chains. The combination of the two can thoroughly open the three-dimensional network structure of the cell wall, maximizing the exposure of internal active ingredients. If the proportion of xylanase is too high, it will introduce small molecule impurities due to excessive degradation of hemicellulose; if the proportion is too low, it will not be able to effectively destroy the cross-linked structure, both of which will reduce the enzymatic hydrolysis efficiency. Preliminary experiments have verified that other conventional enzyme preparations have significantly poor cell wall breaking effects on the Brazilian ginseng-guarana mixed raw material: the substrates of pectinase, protease, and amylase are pectin, protein, and starch, respectively, while the total proportion of these components in the cell wall of Brazilian ginseng and guarana is less than 10%. Such enzyme preparations cannot target the core cellulose-hemicellulose cross-linked structure, and the dissolution rate of active ingredients after enzymatic hydrolysis is not significantly improved. Moreover, the use of pectinase and protease will introduce enzyme protein impurities into the system, which are easy to combine with active ingredients to form complexes, thereby reducing the purity of the subsequently extracted active ingredients. Preliminary experimental data show that the 7:3 cellulase-xylanase combination selected in this invention improves the enzymatic hydrolysis efficiency by 68.5% compared to single cellulase, by 82.3% and 85.7% compared to cellulase-pectinase and cellulase-protease complex systems, respectively, and by 90.2% compared to xylanase-amylase combination. The cell wall breaking and dissolution effect is significantly better than other enzyme preparations and combinations.

[0020] After pretreatment, the present invention further encapsulates the Brazilian ginseng-guarana complex water extract and freeze-dries it to obtain the Brazilian ginseng-guarana encapsulated complex water extract; the encapsulation wall material is a mixture of maltodextrin and β-cyclodextrin, and the mass ratio of maltodextrin to β-cyclodextrin is (7:3)-(9:1).

[0021] The maltodextrin and β-cyclodextrin composite wall material used in this invention forms a core-shell encapsulation structure. β-cyclodextrin has a hydrophobic cavity structure that can specifically encapsulate the steroid nucleus of ecdysterone and embed it inside the cavity. Maltodextrin acts as an outer hydrophilic carrier to form a protective shell. This structure provides the core function of targeted protection for the active ingredients: the cavity of β-cyclodextrin can physically isolate external oxygen molecules from contact with the steroid structure of ecdysterone, effectively preventing oxidative degradation or conformational inversion of the steroid structure; at the same time, in the acidic gastric juice environment (pH 1.0-3.0), the molecular structure of the composite wall material remains stable and will not swell or disintegrate, avoiding the destruction of ecdysterone and caffeine by gastric acid in the gastric juice, thus achieving stable retention of the active ingredients in the gastric juice. In a neutral intestinal fluid environment (pH 7.0-7.4), the composite wall material swells and disintegrates under the influence of intestinal acidity and alkalinity and intestinal enzymes, releasing the encapsulated active ingredients in a targeted manner. This allows the active ingredients to be fully absorbed within the intestinal absorption window, achieving controlled release in the intestine.

[0022] In this invention, the thickness of the Brazilian ginseng slices is preferably 1-3 mm, more preferably 2 mm; the guarana is preferably pulverized to a particle size of 1-5 mm after the brown outer seed coat is removed, more preferably 2-3 mm.

[0023] In this invention, ultrasonic treatment at 250 W and 40 kHz for 10 min is preferred. Ultrasonic treatment utilizes the shock waves and microjets generated by cavitation effect to break down plant cell walls and intercellular matrix, making the cell wall structure loose and porous, exposing the binding sites of active ingredients, which facilitates subsequent enzymatic hydrolysis, while reducing the phenomenon of active ingredients being encapsulated by the cell wall and promoting their full dissolution during water extraction.

[0024] In this invention, it is preferred to add 0.1% of cellulase-xylanase in a mass ratio of 7:3 and enzymatically hydrolyze at 45 °C for 30 min; further preferred inactivation conditions are heating to 85-95 °C and holding for 8-12 min, and most preferably holding at 90 °C for 10 min.

[0025] In this invention, the preferred material-to-liquid ratio for ultrasound-complex enzyme pretreatment is 1 g:30-50 mL, more preferably 1 g:40 mL, and even more preferably the pH of the material solution is adjusted to 4.5-5. As one possible implementation, citric acid / sodium bicarbonate is used for adjustment.

[0026] In this invention, preferably, water is added to 100-133 times the total mass of the mixed raw materials during extraction, the extraction time is 30-90 minutes, and the extraction is performed 1-3 times; more preferably, water is added to 133 times the total mass of the mixed raw materials, the extraction time is 60 minutes, and the extraction is performed once. This combination of parameters ensures sufficient dissolution of active ingredients while balancing extraction efficiency and production costs, and avoids over-extraction that leads to increased impurities.

[0027] In this invention, after extraction, the solution is preferably filtered through 200-300 mesh gauze to collect the filtrate and remove solid impurities. More preferably, it is filtered through 250 mesh gauze. The mixed filtrate is preferably concentrated to 5%-25% of its original volume, more preferably 10%-20%, and even more preferably 15%.

[0028] In this invention, the preferred mass fraction of the encapsulated wall material aqueous solution is 10%-15%, more preferably 12%; the preferred volume ratio of the Brazilian ginseng-guarana composite water extract to the wall material aqueous solution is 1:2-1:4, more preferably 1:3.

[0029] In this invention, the preferred freeze-drying process includes: pre-freezing the encapsulated mixture to -40 to -60 °C, more preferably -50 °C, for 4-6 h, more preferably 5 h, followed by freeze-drying at a vacuum of 10-30 Pa and a drying temperature of -20 to -10 °C for 24-36 h, more preferably at 20 MPa and -15 °C for 30 h; the resulting powder has a particle size of 20-80 μm and an encapsulation rate of ≥85%.

[0030] This invention also provides the Brazilian ginseng-guarana encapsulated water extract prepared by the above method and its application in the preparation of anti-fatigue functional beverages.

[0031] This invention also provides an anti-fatigue functional beverage, comprising the following components by mass concentration: 25%-35% of the above-mentioned Brazilian ginseng-guarana encapsulated complex water extract, 3%-4% maltitol, 0.01%-0.02% citric acid, with the balance being water; preferably, 30% Brazilian ginseng-guarana encapsulated complex water extract, 4% maltitol, 0.01% citric acid, with the balance being water. This formulation ensures that the concentration of active ingredients in the beverage meets the standard (≥0.2 mg / mL), and also allows for taste adjustment through maltitol and citric acid, thus meeting the consumption requirements of the beverage.

[0032] The technical solutions provided by the present invention will be described in detail below with reference to the embodiments, but they should not be construed as limiting the scope of protection of the present invention.

[0033] Example 1 A Brazilian ginseng-guarana encapsulated water extract is prepared as follows: (1) Raw material mixing: Weigh 15g of Brazilian ginseng (slice thickness of 1-3 mm) and 135g of guarana (particle size of 1-5 mm) at a mass ratio of 1:9, place them in a stainless steel mixing container, stir for 5 min until they are evenly mixed, and obtain 150g of mixed raw materials. (2) Ultrasonic-compound enzyme pretreatment: Add 6000 mL of deionized water to the mixed raw materials to make the material-liquid ratio 1 g: 40 mL, and adjust the pH of the system to 4.8 with citric acid aqueous solution; place the mixing tank in the ultrasonic equipment, set the ultrasonic power to 250 W and the frequency to 40 kHz, and perform continuous ultrasonic treatment for 10 min; after the ultrasonic treatment, add 0.15 g of compound enzyme (the mass ratio of cellulase to xylanase is 7:3) to the material, and the total amount of compound enzyme added is 0.1% of the total mass of the mixed raw materials; transfer the mixing tank to a 45℃ constant temperature water bath, keep warm for 30 min for enzymatic hydrolysis, and after the enzymatic hydrolysis is completed, raise the temperature to 90 ℃ and keep warm for 10 min to achieve enzyme inactivation; (3) Extraction: Add deionized water to the pretreated material to 13950 mL, so that the total amount of water added reaches 133 times the total mass of the mixed raw materials. Then raise the temperature to 95 °C and extract at a constant temperature for 60 min. The extraction is performed once. After extraction, filter with 250 mesh gauze, collect the filtrate and remove solid impurities. (4) Concentration: Transfer the collected filtrate to a vacuum concentrator, set the concentration temperature to 50 ℃ and the vacuum degree to -0.085 MPa, and carry out vacuum concentration until the volume of the concentrated liquid reaches 25% of the original filtrate volume. Stop the concentration to obtain Brazilian ginseng-guarana complex water extract. (5) Encapsulation: Weigh maltodextrin and β-cyclodextrin at a mass ratio of 8:2, mix them evenly, add deionized water, place them on a digital display stirrer and stir to dissolve, and prepare a wall material aqueous solution with a mass fraction of 12%; mix the Brazilian ginseng-guarana composite water extract with the wall material aqueous solution at a volume ratio of 1:3, turn on the digital display stirrer and stir at a speed of 500 r / min for 10 min to make the two fully mixed evenly; (6) Freeze-drying: Pour the above mixture into the tray of the freeze dryer and spread it to a thickness of 6 mm. First, place the tray in the freeze dryer and pre-freeze it to -50 ℃ for 5 h. Then, adjust the vacuum degree of the freeze dryer to 20 Pa and the drying temperature to -15 ℃. Freeze-dry under these conditions for 30 h. After drying, pass it through an 80-mesh sieve and collect the powdery product at the outlet, which is the Brazilian ginseng-guarana encapsulated composite water extract.

[0034] Example 2 A Brazilian ginseng-guarana encapsulated water extract is prepared as follows: (1) Raw material mixing: Weigh 15g of Brazilian ginseng (slice thickness 1-3 mm) and 135g of guarana (particle size 1-5 mm) at a mass ratio of 1:9, place them in a stainless steel mixing container, stir for 5 min until they are evenly mixed, and obtain 150g of mixed raw materials. (2) Ultrasonic-compound enzyme pretreatment: Add 4500 mL of deionized water to the mixed raw materials to make the material-liquid ratio 1 g:30 mL, and adjust the pH of the system to 4.5 with citric acid aqueous solution; place the mixing tank in the ultrasonic equipment, set the ultrasonic power to 200 W and the frequency to 35 kHz, and perform continuous ultrasonic treatment for 5 min; after the ultrasonic treatment, add 0.12 g of compound enzyme (the mass ratio of cellulase to xylanase is 6:4) to the material, and the total amount of compound enzyme added is 0.08% of the total mass of the mixed raw materials; transfer the mixing tank to a 40℃ constant temperature water bath, keep it warm for 20 min for enzymatic hydrolysis, and after the enzymatic hydrolysis is completed, raise the temperature to 85 ℃ and keep it warm for 8 min to achieve enzyme inactivation; (3) Extraction: Add deionized water to the pretreated material to 10,500 mL, so that the total amount of water added is 100 times the total mass of the mixed raw materials. Then heat to 90 °C and extract at a constant temperature for 30 min. The extraction is performed once. After extraction, filter with 200 mesh gauze, collect the filtrate and remove solid impurities. (4) Concentration: Transfer the collected filtrate to a vacuum concentrator, set the concentration temperature to 45 ℃ and the vacuum degree to -0.08 MPa, and carry out vacuum concentration until the volume of the concentrated liquid reaches 5% of the original filtrate volume. Stop the concentration to obtain Brazilian ginseng-guarana complex water extract. (5) Encapsulation: Weigh maltodextrin and β-cyclodextrin at a mass ratio of 7:3, mix them evenly, add deionized water, place them on a digital display stirrer and stir to dissolve, and prepare a wall material aqueous solution with a mass fraction of 10%; mix the Brazilian ginseng-guarana composite water extract with the wall material aqueous solution at a volume ratio of 1:2, turn on the digital display stirrer and stir at a speed of 500 r / min for 10 min to make the two fully mixed evenly; (6) Freeze-drying: Pour the above mixture into the tray of the freeze dryer and spread it to a thickness of 5 mm. First, place the tray in the freeze dryer and pre-freeze it to -40 ℃ for 4 h. Then, adjust the vacuum degree of the freeze dryer to 10 Pa and the drying temperature to -20 ℃. Freeze-dry under these conditions for 24 h. After drying, pass it through an 80-mesh sieve and collect the powdery product at the outlet, which is the Brazilian ginseng-guarana encapsulated composite water extract.

[0035] Example 3 A Brazilian ginseng-guarana encapsulated water extract is prepared as follows: (1) Raw material mixing: Weigh 15 g of Brazilian ginseng (slice thickness of 1-3 mm) and 135 g of guarana (particle size of 1-5 mm) at a mass ratio of 1:9, place them in a stainless steel mixing container, stir for 5 min until they are evenly mixed, and obtain 150 g of mixed raw materials. (2) Ultrasonic-compound enzyme pretreatment: Add 7500 mL of deionized water to the mixed raw materials to make the material-liquid ratio 1 g: 50 mL, and adjust the pH of the system to 5.0 with citric acid aqueous solution; place the mixing tank in the ultrasonic equipment, set the ultrasonic power to 300 W and the frequency to 45 kHz, and perform continuous ultrasonic treatment for 15 min; after the ultrasonic treatment, add 0.18 g of compound enzyme (the mass ratio of cellulase to xylanase is 8:2) to the material, and the total amount of compound enzyme added is 0.12% of the total mass of the mixed raw materials; transfer the mixing tank to a 50℃ constant temperature water bath, keep it warm for 40 min for enzymatic hydrolysis, and after the enzymatic hydrolysis is completed, raise the temperature to 95 ℃ and keep it warm for 12 min to achieve enzyme inactivation; (3) Extraction: Add deionized water to the pretreated material to 12450 mL, so that the total water added reaches 133 times the total mass of the mixed raw materials. Then raise the temperature to 100℃ and extract at a constant temperature for 90 min. The extraction is repeated 3 times. After each extraction, filter with 300 mesh gauze, collect the filtrates and combine them. (4) Concentration: Transfer the combined filtrate to a vacuum concentrator, set the concentration temperature to 60 ℃ and the vacuum degree to -0.09 MPa, and carry out vacuum concentration until the volume of the concentrated liquid reaches 25% of the original filtrate volume. Stop the concentration to obtain Brazilian ginseng-guarana complex water extract. (5) Encapsulation: Weigh maltodextrin and β-cyclodextrin at a mass ratio of 9:1, mix them evenly, add deionized water, place them on a digital display stirrer and stir to dissolve, and prepare a wall material aqueous solution with a mass fraction of 15%; mix the Brazilian ginseng-guarana composite water extract with the wall material aqueous solution at a volume ratio of 1:4, turn on the digital display stirrer and stir at a speed of 500 r / min for 10 min to make the two fully mixed evenly; (6) Freeze-drying: Pour the above mixture into the tray of the freeze dryer and spread it to a thickness of 8 mm. First, place the tray in the freeze dryer and pre-freeze it to -60 ℃ for 6 h. Then, adjust the vacuum degree of the freeze dryer to 30 Pa and the drying temperature to -10 ℃. Freeze-dry under these conditions for 36 h. After drying, pass it through an 80-mesh sieve and collect the powdery product at the outlet, which is the Brazilian ginseng-guarana encapsulated complex water extract.

[0036] Example 4 An anti-fatigue functional beverage, comprising the following ingredients by mass concentration: Example 1: Brazilian ginseng-guarana encapsulated water extract 30%, maltitol 4%, citric acid 0.01%, balance being purified water.

[0037] Example 5 An anti-fatigue functional beverage, comprising the following ingredients by mass concentration: Example 2: 25% Brazilian ginseng-guarana encapsulated water extract, 3% maltitol, 0.02% citric acid, with the remainder being purified water.

[0038] Example 6 An anti-fatigue functional beverage, comprising the following ingredients by mass concentration: Example 3: Brazilian ginseng-guarana encapsulated water extract 35%, maltitol 3.5%, citric acid 0.015%, balance being purified water.

[0039] Experimental Example 1 I. Experimental Design The single-factor variable method was used to set up 4 groups of experiments. The raw material amount, extraction process and concentration process of each group were completely the same, and only the pretreatment method was different. Each group was repeated 3 times in parallel.

[0040] 1. Group settings Control group (no pretreatment): Mixed raw materials + 133 times water → extraction at 95 ℃ for 60 min → filtration → concentration; Experimental Group 1 (Ultrasonic Pretreatment): Mixed raw materials → Add water at a ratio of 1:40 → Ultrasonic treatment (250 W, 40 kHz, 10 min, liquid temperature ≤50 ℃) → 133 times water → Extraction at 95 ℃ for 60 min → Filtration → Concentration; Experimental Group 2 (Enzyme Pretreatment): Mix raw materials → Add water at a material-to-liquid ratio of 1:40 → Adjust pH to 4.8 → Add compound enzyme (cellulase: xylanase = 7:3, total addition 0.1%) → Enzymatic hydrolysis at 45℃ for 30 min → Inactivation at 90℃ for 10 min → Add water to 133 times → Extract at 95℃ for 60 min → Filter → Concentrate; Experimental Group 3 (Ultrasound-compound enzyme pretreatment): Mix raw materials → Add water at a material-to-liquid ratio of 1:40 → Adjust pH to 4.8 → Ultrasound treatment (250 W, 40 kHz, 10 min) → Add compound enzyme (cellulase: xylanase = 7:3, total addition 0.1%) → Enzymatic hydrolysis at 45℃ for 30 min → Inactivation at 90℃ for 10 min → Add water to 133 times → Extract at 95℃ for 60 min → Filter → Concentrate.

[0041] Raw material dosage: Each group consists of 15g of Brazilian ginseng and 135g of guarana, with a total mass of 150g after mixing; Concentration conditions: Concentrate under reduced pressure (50 ℃, -0.085 MPa) to 25% of the original filtrate volume to obtain a compound water extract, which is then stored in a refrigerator at 4 ℃ for later use.

[0042] II. Indicator Detection Methods 1. Determination of active ingredient dissolution (UPLC method) Chromatographic conditions: Waters ACQUITY UPLC HSS T3 column (2.1 mm × 100 mm, 1.8 μm); mobile phase A (0.1% formic acid in water), B (0.1% formic acid in acetonitrile), gradient elution (0 min: 98% A / 2% B; 1 min: 90% A / 10% B; 5 min: 90% A / 10% B; 8 min: 80% A / 20% B; 10 min: 2% A / 98% B; 14 min: 2% A / 98% B; 15 min: 98% A / 2% B); flow rate 0.4 mL / min; column temperature 40 ℃; injection volume 1 μL; detection wavelength 270 nm (caffeine), 242 nm (ecdysterone).

[0043] Standard curve plotting: Prepare precise caffeine standard solutions (concentration 7.8125-500 μg / mL) and ecdysterone standard solutions (concentration 7.8125-500 μg / mL). Inject them under the chromatographic conditions described above. Plot a standard curve using peak area (Y) against concentration (X) to obtain the regression equation: Caffeine: Y = 4241.6X - 8626.7, R 2 = 0.9996; Ecdysterone: Y = 2107.7X + 5953.7, R 2 = 0.9998.

[0044] Sample testing: Take the concentrated compound water extracts from each group, dilute them 10 times with methanol, filter them through a 0.22 μm filter membrane, and inject them into the solution. Calculate the caffeine and ecdysterone content according to the regression equation, and multiply them by the volume of the concentrated solution to obtain the total dissolution amount.

[0045] 2. Storage stability testing Sample processing: The composite water extracts of each group were stored in a constant temperature and humidity chamber (25 ℃, 60% relative humidity) for 6 months; Detection method: Following the "Active Ingredient Dissolution Detection" method, the content of active ingredients was measured before storage and after 6 months of storage, and the retention rate was calculated. Retention rate (%) = (content after 6 months of storage / content before storage) × 100%.

[0046] III. Data Statistical Methods Data analysis was performed using SPSS 26.0 statistical software. All data are expressed as mean ± standard deviation (x ± SD). Normality tests (Shapiro-Wilk test) and homogeneity of variance tests (Lveneer test) were performed first. After confirming normality and homogeneity of variance, one-way ANOVA was used. Multiple comparisons between groups were performed using the LSD method. P <0.05 indicates that the difference is statistically significant.

[0047] IV. Test Results 1. Effects of different pretreatment methods on the dissolution rate of active ingredients As shown in Table 1, when ultrasound pretreatment or enzyme pretreatment was used alone, the dissolution of caffeine and ecdysterone increased by 32%-40% compared with the control group, but there was no significant difference between the groups. P >0.05); while the dissolution rate of the ultrasound-composite enzyme synergistic pretreatment (experimental group 3) was more than 78% higher than that of the control group, and significantly higher than that of the single pretreatment group ( P The result was <0.01, demonstrating a synergistic effect between ultrasound and the complex enzyme, which can more thoroughly break down plant cell walls and promote the release of active ingredients.

[0048] Table 1. Effects of different pretreatment methods on the dissolution rate of active ingredients.

[0049] Note: Compared with the control group Compared with the control group P <0.01; △△ Compared with experimental group 1 and experimental group 2 P <0.01; the same as in the table below.

[0050] 2. The impact of different pretreatment methods on storage stability As shown in Table 2, the mean retention rate of experimental group 3 at 6 months reached 95.8%, which was 26% higher than that of the control group and 17%-18% higher than that of the single pretreatment group. P The value <0.01 indicates that synergistic pretreatment can reduce the encapsulation and polymerization of active ingredients by macromolecular impurities (such as cellulose and pectin), thereby improving storage stability.

[0051] Table 2. Impact of different pretreatment methods on storage stability

[0052] Experimental Example 2 I. Experimental Design Wall material selection test and drying parameter optimization test were conducted separately, with each group repeated 3 times in parallel. The core material dosage and embedding process (except for variables) were completely consistent.

[0053] (a) Wall material selection test Total wall material usage = total mass of active ingredients in the core material × 6; wall material aqueous solution mass fraction 12%; volume ratio of composite water extract to wall material aqueous solution 1:3; freeze drying; Group settings: Control group 1: Single wall material - maltodextrin; Control group 2: Single wall material - β-cyclodextrin; Control group 3: Composite wall material - maltodextrin: gum arabic = 7:3; Experimental group: Composite wall material - maltodextrin: β-cyclodextrin = 8:2.

[0054] (II) Optimization test of freeze-drying parameters With freeze-drying process parameters as variables, the wall material combination (maltodextrin:β-cyclodextrin=8:2), the total amount of wall material (=total mass of active ingredients in the core material x6 times), and the volume ratio of the composite water extract to the wall material aqueous solution (1:3) were all kept constant.

[0055] Group settings: Experimental group A: Pre-freezing temperature -50 ℃ (pre-freezing for 5 h), followed by freeze-drying at a vacuum of 20 Pa and a drying temperature of -15 ℃ for 30 h.

[0056] Experimental Group B: Pre-freezing temperature -30 ℃ (pre-freezing for 5 h), drying conditions the same as Experimental Group A.

[0057] Experimental Group C: Pre-freezing conditions were the same as those of Experimental Group A, and drying conditions were vacuum degree of 20 Pa, drying temperature of -5 ℃, and drying time of 30 h.

[0058] Experimental Group D: Pre-freezing and drying temperature conditions were the same as in Experimental Group A, but the drying time was shortened to 18 hours.

[0059] II. Indicator Detection Methods 1. Embedding rate detection Total active ingredient content determination: Take 0.1 g of powder, add 10 mL of methanol, extract by ultrasonication for 30 min (250 W, 40 kHz), centrifuge at 12000 r / min for 10 min, take the supernatant and determine the total active ingredient content by UPLC method according to Example 1; Detection of surfactant content: Take 0.1 g of powder, add 10 mL of deionized water, stir at room temperature for 10 min (500 r / min), centrifuge at 12000 r / min for 10 min, take the supernatant and detect the surfactant content by UPLC method; Encapsulation rate calculation: Encapsulation rate (%) = (W 总 -W 表 ) / W 总×100%.

[0060] 2. Storage stability test Sample preparation: Store the powder in a constant temperature and humidity chamber (25 ℃, 60% relative humidity) for 6 months; Detection method: The total active ingredient content was detected before storage and after 6 months of storage (according to the "Total Active Ingredient Content Detection Method"), and the retention rate was calculated. Retention rate (%) = (Total content after 6 months of storage / Total content before storage) × 100%.

[0061] 3. Solubility test Sample preparation: Take 1 g of powder, add 10 mL of deionized water (25 ℃), and stir at 500 r / min for 10 min; Detection method: Take the supernatant, filter it through a 0.22 μm filter membrane, and determine the content of active ingredients by UPLC method. Calculate the solubility rate. Dissolution rate (%) = (Total amount of active ingredients in the supernatant / Total amount of active ingredients added to the sample) × 100%.

[0062] 4. Powder morphology and particle size detection Particle size detection: Take an appropriate amount of powder, dilute it with deionized water, and then use a laser particle size analyzer to detect the D50 particle size distribution.

[0063] 5. Detection of active ingredient loss rate Calculation method: Active ingredient loss rate (%) = (Total mass of active ingredients in the core material before embedding - Total mass of active ingredients in the powder after embedding) / Total mass of active ingredients in the core material before embedding × 100%.

[0064] III. Data Statistical Methods Same as Experiment 1 (SPSS 26.0, one-way ANOVA, LSD multiple comparisons). P <0.05 indicates a significant difference.

[0065] IV. Test Results 1. Wall material selection test As shown in Table 3, the composite wall material (maltodextrin:β-cyclodextrin = 8:2) of this invention achieves an encapsulation rate of 92.3%, significantly higher than that of single wall materials (72.5%-88.7%) and other composite wall materials (78.3%). Simultaneously, it balances high stability and good solubility, solving the problems of low encapsulation rate of single maltodextrin and poor solubility of single β-cyclodextrin. Clearly, ordinary maltodextrin lacks a specific cavity structure and only provides physical encapsulation, leading to rapid dissolution in acidic gastric juice. This results in premature release of the encapsulated active ingredient in gastric juice, causing not only the destruction of the steroidal structure of ecdysterone by gastric acid and rapid decomposition of caffeine, but also significantly reducing bioavailability because the active ingredient misses the optimal absorption window in the intestine. The composite wall material encapsulation design of this invention structurally solves this technical problem.

[0066] Table 3. Effects of different wall materials on embedding effect

[0067] 2. Parameter optimization experiment As shown in Table 4, the embedding effect was best and the active ingredient loss rate was lowest when using the drying and freezing parameters of experimental group A. Insufficient pre-freezing temperature (experimental group B, -30 ℃) resulted in insufficient crystallization or partial melting of the sample, causing structural collapse and exposure of active ingredients during subsequent drying, thus significantly reducing the embedding rate and increasing the loss rate. Excessively high drying temperature (experimental group C, -5 ℃) caused the ice crystal sublimation rate to be too fast, damaging the integrity of the embedding structure and affecting the embedding effect. Insufficient drying time (experimental group D, 18 h) resulted in residual moisture, making the powder prone to clumping, and the residual moisture may promote the degradation of active ingredients, leading to an increased loss rate.

[0068] Table 4. Effects of different parameters on embedding effect

[0069] Experimental Example 3 I. Experimental Materials Core material (unencapsulated water extract): Brazilian ginseng-guarana complex water extract (concentrated to 25% of the original volume) was prepared according to steps (1)-(4) of Example 1 and stored at 4°C for later use.

[0070] Encapsulated product: Prepare Brazilian ginseng-guarana encapsulated composite water extract powder (maltodextrin:β-cyclodextrin=8:2) according to the complete steps (1)-(6) of Example 1, and store it in a sealed, light-proof, and room-temperature environment for later use.

[0071] Simulated gastric juice: Take 2.0 g of NaCl, add 7.0 mL of concentrated hydrochloric acid, and dilute to 1000 mL with deionized water. Adjust the pH to 1.2±0.1, and add pepsin (activity units ≥250 NFU / mg) to make the final concentration 3.2 mg / mL. Prepare fresh before use.

[0072] Simulated intestinal fluid: Take 6.8 g of KH2PO4, add 77 mL of 0.2 mol / L NaOH solution, bring the volume to 1000 mL with deionized water, adjust the pH to 7.4±0.1, add pancreatic enzyme (activity ≥4000 NFU / mg) to make the final concentration 10 mg / mL, and prepare fresh before use.

[0073] II. Experimental Design Two groups were set up, with equal amounts of caffeine and ecdysterone, and each group was repeated 3 times in parallel.

[0074] Control group: No water extract encapsulated; Experimental group: The composite water extract powder was encapsulated and reconstituted with deionized water.

[0075] Gastrointestinal sequential release assay procedure: (1) Simulated gastric juice stage: Each group of samples was added to 50 mL of simulated gastric juice and incubated in a 37℃ constant temperature water bath shaker (100 r / min) for 2 h to simulate the gastric digestion process; 2 mL samples were taken at 0.5 h, 1 h and 2 h respectively, and 1 mL of 0.5 mol / L NaOH solution was added immediately to terminate the enzymatic reaction. The samples were centrifuged at 12000 r / min for 10 min and the supernatant was taken for testing.

[0076] (2) Simulated intestinal fluid stage: After the gastric fluid stage incubation is completed, add an equal volume (50 mL) of simulated intestinal fluid (adjust the pH of the mixed system to about 7.2-7.4) to the remaining reaction solution, and continue to incubate in a 37℃ constant temperature water bath shaker (100 r / min) for 2 h; take 2 mL samples at 0.5 h, 1 h and 2 h respectively, add 1 mL of methanol immediately to terminate the enzymatic hydrolysis reaction, centrifuge at 12000 r / min for 10 min, and take the supernatant for testing.

[0077] III. Indicator Detection Methods Active ingredient content detection: The contents of caffeine and ecdysterone in the supernatant of each sample at each time point were determined according to the method of Experiment Example 1.

[0078] Calculation indicators: (1) Gastric juice retention rate: Gastric juice retention rate (%) = active ingredient content at each time point in the gastric juice stage / total amount of initial active ingredient × 100%; (2) Cumulative release rate in intestinal fluid stage: Cumulative release rate in intestinal fluid (%) = Content of active ingredient in supernatant at each time point in the intestinal fluid stage / Total amount of initial active ingredient × 100%; (3) Loss rate of active ingredients in gastric juice stage: Gastric juice loss rate (%) = 100% - 2 h retention rate in gastric juice stage (%).

[0079] IV. Data Statistical Methods Same as Experiment 1 (SPSS 26.0, one-way ANOVA, LSD multiple comparisons). P <0.05 indicates a significant difference.

[0080] V. Test Results As shown in Table 5, the active ingredients in the unencapsulated water extract degraded significantly in simulated gastric juice. After 2 hours, the retention rates of caffeine and ecdysterone were only 81.3% and 72.6%, respectively, indicating that the steroidal structure of ecdysterone is prone to conformational changes or ring-opening degradation under strong acid conditions. The 2-hour retention rates of caffeine and ecdysterone in the encapsulated product of this invention were 96.8% and 95.4%, respectively, significantly higher than those in the control group. P The value <0.01 indicates that the composite wall material forms an effective protective barrier in the gastric acid environment. The hydrophobic cavity of β-cyclodextrin encapsulates and isolates the steroidal core of ecdysterone, while the outer layer of maltodextrin further blocks gastric acid penetration, achieving stable retention of the active ingredient in gastric juice. Upon entering the simulated intestinal fluid stage, the active ingredient in the encapsulated product of this invention exhibits sustained-release characteristics: a cumulative release rate of 42.5%-43.8% at 0.5 h, 71.6%-73.2% at 1 h, and 90.1%-91.5% at 2 h, with a smooth and continuous release curve. In contrast, the unencapsulated water extract, because the active ingredient exists in a free state, dissolves almost instantaneously in intestinal juice, achieving a cumulative release rate of over 85% at 0.5 h and nearly 100% at 2 h. The sustained-release characteristics of the encapsulated product ensure continuous and uniform release of the active ingredient within the intestinal absorption window, facilitating full absorption by the intestinal mucosa and preventing absorption saturation or rapid metabolic clearance due to excessively high local concentrations of the free active ingredient, thereby improving bioavailability.

[0081] The encapsulated product of this invention exhibits a total active ingredient retention rate of ≥95% in the gastric juice stage (2 hours) and a cumulative release rate of ≥90% in the intestinal juice stage (2 hours). In contrast, the unencapsulated water extract suffers a 18%-27% loss of active ingredients in the gastric juice stage. Although it is rapidly released in the intestinal juice stage, due to the loss in the gastric juice stage and the lack of a sustained-release mechanism, the total amount of effective active ingredients that can actually be absorbed by the intestine is significantly lower than that of the encapsulated product.

[0082] Table 5. Retention and cumulative release rate of active ingredients during simulated gastric and intestinal fluid stages.

[0083] Note: Compared with the control group at the same time point P <0.01.

[0084] Test Example 4 1. Test materials Test samples: The beverage of the present invention (30% encapsulated composite aqueous extract powder + 3.5% maltitol + 0.015% citric acid + deionized water), control anti-fatigue beverage (prepared according to the optimal process of CN 113229500 A); Experimental animals: 48 SPF-grade male Kunming mice, weighing 18 - 22 g, purchased from Beijing Huafukang Biotechnology Co., Ltd., license number: SCXK(Beijing)2020 - 0004; Feeding conditions: SPF-grade animal room (temperature 22 ± 2 °C, humidity 50 ± 5%, 12 h light / 12 h darkness), free access to food and water, and the experiment started after 3 days of adaptive feeding.

[0085] 3. Experimental design (1) Grouping setting The 48 mice were randomly divided into 4 groups, with 12 mice in each group, and gavaged continuously for 35 days: Blank control group: gavaged with sterile normal saline daily, gavage volume 10 mL / kgBW; Unencapsulated anti-fatigue beverage group: gavaged with the control anti-fatigue beverage daily, gavage volume 10 mL / kgBW; Low-dose group of the beverage of the present invention: gavaged with the beverage of Example 4 of the present invention daily, gavage volume 5 mL / kgBW; High-dose group of the beverage of the present invention: gavaged with the beverage of Example 4 of the present invention daily, gavage volume 10 mL / kgBW.

[0086] (2) Preparation of test samples Beverage of the present invention: Weigh 30 g of the encapsulated composite aqueous extract powder prepared in Example 1, 3.5 g of maltitol, and 0.015 g of citric acid, add deionized water to 100 g, stir for 10 min until completely dissolved, refrigerate at 4 °C for later use, and restore to room temperature before use; Unencapsulated anti-fatigue beverage: Prepared according to the optimal process of CN 113229500 A, refrigerated at 4 °C for later use, and restored to room temperature before use.

[0087] 4. Index detection methods (1) Detection of load-bearing swimming time Detection time: 30 min after the last gavage; Detection method: A lead wire with a load of 5% of the body weight is tied to the root of the mouse's tail, and the mouse is placed in a swimming tank (water depth 30 cm, water temperature 25 ± 1 °C), and the time from the start of swimming to drowning death of the mouse (i.e., the load-bearing swimming time) is recorded.

[0088] (2) Detection of serum urea content Detection time: 30 min after the last gavage; Treatment method: Mice swam in 30 ℃ water for 90 min, rested for 60 min, and 0.5 mL of blood was collected from the eyeballs. The blood was placed in a 4 ℃ refrigerator and allowed to stand for 3 h until clotting. The blood was then centrifuged at 3000 r / min for 15 min to separate the serum. Detection method: Follow the instructions for the urea test kit and determine the serum urea content on a fully automated biochemical analyzer.

[0089] (3) Detection of area under the blood lactate curve Testing time: 30 minutes after the last gavage; Blood collection time points: before swimming (resting blood), 0 min after swimming (blood collection immediately after swimming in 30℃ water for 10 minutes without weight-bearing), and 20 min after swimming (blood collection after resting for 20 minutes). Blood collection method: 10 μL of blood was collected from the inner canthal venous plexus of the eye and added to a reaction tube containing anticoagulant. The tube was then shaken thoroughly. Detection method: Follow the instructions of the lactate test kit and measure the blood lactate concentration using a lactate analyzer; Area under the curve calculation: Blood lactate area under the curve = 5 × (blood lactate value before swimming + 3 × blood lactate value at 0 min after swimming + 2 × blood lactate value at 20 min after swimming).

[0090] (4) Mouse weight monitoring Monitoring time: before the start of the experiment (initial body weight), once a week during gavage, and after the end of the experiment (final body weight). Monitoring method: The weight of mice was measured using an electronic balance, and the data was recorded.

[0091] 5. Data Statistical Methods SPSS 26.0 statistical software was used, and the data are expressed as mean ± standard deviation (x ± SD). Normality and homogeneity of variance tests were performed first. If the requirements were met, one-way ANOVA was used, and the LSD test was used for multiple comparisons between groups. If the data did not conform to a normal distribution or had unequal variances, the rank-sum test was used. P <0.05 indicates that the difference is statistically significant.

[0092] 6. Test Results As shown in Table 6, both the low- and high-dose groups of the beverage of this invention significantly prolonged the weight-bearing swimming time of mice, reduced serum urea levels, and decreased the area under the blood lactate curve (AUC). P <0.05); among them, all indicators of the high-dose group were significantly better than those of the unencapsulated anti-fatigue beverage group (P<0.05), proving that the anti-fatigue effect of the beverage of the present invention is superior.

[0093] In addition, there were no significant differences in initial body weight, final body weight, and weight gain among the groups of mice. P>0.05), proving that the beverage of the present invention has no adverse effects on the growth and development of mice and has good safety.

[0094] Table 6. Effects of different beverages on anti-fatigue related indicators in mice.

[0095] Note: Compared with the blank control group Compared with the blank control group P <0.01; △ Compared with the unmodified anti-fatigue beverage group P <0.05.

[0096] The above description is only a preferred embodiment of the present invention. It should be noted that for those skilled in the art, several improvements and modifications can be made without departing from the principle of the present invention, and these improvements and modifications should also be considered within the scope of protection of the present invention.

Claims

1. A method for preparing a Brazilian ginseng-guarana encapsulated composite water extract, characterized in that, Includes the following steps: Brazilian ginseng and guarana were mixed at a mass ratio of 1:9, and water was added for ultrasonic-compound enzyme pretreatment. After pretreatment, the mixture was extracted and concentrated to obtain a Brazilian ginseng-guarana compound water extract. The ultrasound-composite enzyme pretreatment includes: ultrasound treatment at 200-300 W and 35-45 kHz for 5-15 min, followed by the addition of 0.08%-0.12% of cellulase-xylanase at a mass ratio of (6:4)-(8:2), enzymatic hydrolysis at 40-50 ℃ for 20-40 min, and inactivation. Brazilian ginseng-guarana complex water extract was encapsulated and freeze-dried to obtain Brazilian ginseng-guarana encapsulated complex water extract; the encapsulation wall material was a mixture of maltodextrin and β-cyclodextrin, and the mass ratio of maltodextrin to β-cyclodextrin was (7:3)-(9:1).

2. The preparation method according to claim 1, characterized in that, The Brazilian ginseng slices are 1-3 mm thick, and the guarana is pulverized to a particle size of 1-5 mm after the brown outer seed coat is removed.

3. The preparation method according to claim 1, characterized in that, The material-to-liquid ratio for the ultrasound-composite enzyme pretreatment is 1g:30-50mL.

4. The preparation method according to claim 1, characterized in that, During the extraction, water is added to 100-133 times the total mass of the mixed raw materials, the extraction time is 30-90 minutes, and the extraction is performed 1-3 times.

5. The preparation method according to claim 1, characterized in that, After extraction, the mixture is filtered and concentrated to 5%-25% of its original volume.

6. The preparation method according to claim 1, characterized in that, The encapsulated wall material aqueous solution has a mass fraction of 10%-15%, and the volume ratio of the Brazilian ginseng-guarana composite water extract to the wall material aqueous solution is 1:2-1:

4.

7. The preparation method according to claim 1, characterized in that, The freeze-drying process includes: pre-freezing the encapsulated mixture to -40 to -60 °C for 4-6 h, followed by freeze-drying at a vacuum of 10-30 Pa and a drying temperature of -20 to -10 °C for 24-36 h. The resulting powder has a particle size of 20-80 μm and an encapsulation rate of ≥85%.

8. The Brazilian ginseng-guarana encapsulated composite water extract prepared by the preparation method according to any one of claims 1-7.

9. The application of the preparation method according to any one of claims 1-7 or the Brazilian ginseng-guarana encapsulated complex water extract according to claim 8 in the preparation of an anti-fatigue functional beverage.

10. A fatigue-relieving functional beverage, characterized in that, The composition by mass concentration includes the following components: 25%-35% of the Brazilian ginseng-guarana encapsulated complex water extract as described in claim 8, 3%-4% maltitol, 0.01%-0.02% citric acid, and the balance being water.

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