A composition for alleviating physical fatigue and a method of preparing the same

Abstract

The application discloses a kind of relieving physical fatigue compositions and preparation method thereof, belong to functional food and enzyme engineering technical field;For the technical contradiction that tannic acid astringency is heavy in existing Paraguay tea beverage, and acidic environment is easy to lead to ginsenoside Rb1 hydrolysis inactivation, a kind of step temperature-pH coupling immobilized enzyme catalysis synergistic treatment method is proposed;The method first uses C8-C18 alkyl hydrophobic modification mesoporous silica fixed tannin enzyme, under pH 5.0-6.0, 40-50 DEG C, high-efficiency astringency is removed;Subsequently under the condition that enzyme preparation is not separated, in situ pH is adjusted to 6.5-7.0 and temperature is reduced to 30-35 DEG C, then ginseng extract is added to carry out low-temperature protection reaction, and the destruction of ginsenoside Rb1 glycosidic bond by acid / heat is avoided from the root.The experiment shows that the application can make tannic acid removal rate ≥72.5%, ginsenoside Rb1 retention rate ≥82.5%, and caffeine retention rate ≥86.9%, which is significantly better than traditional process.

Description

Technical Field

[0001] This invention relates to the fields of functional foods and enzyme engineering technology, specifically to a composition for relieving physical fatigue and its preparation method. Background Technology

[0002] In recent years, with the rapid development of the sports nutrition and functional beverage market, anti-fatigue drinks with Paraguayan tea (yerba mate) and ginseng extract as core active ingredients have attracted much attention. Yerba mate is rich in caffeine, polyphenols, and saponins, and has significant effects in refreshing the mind and anti-oxidation. Ginsenoside Rb1 reduces oxidative stress in skeletal muscle by activating the PI3K / Akt / Nrf2 signaling pathway, which is the core material basis for its anti-fatigue effect. However, the high content of tannins in yerba mate leads to a rough and bitter taste, which seriously restricts the sensory acceptance of its processed products. At the same time, the tetrasaccharide chain structure at the C20 position of ginsenoside Rb1 is easily hydrolyzed into ginsenoside Rd in acidic environments or under heating conditions, resulting in a significant decrease in anti-fatigue activity. This contradiction between "removing astringency and preserving efficacy" has become a technical bottleneck restricting the development of high-quality anti-fatigue products.

[0003] Chinese patent application CN117137141A discloses a composition for combating exercise-induced fatigue and improving hypoxia tolerance, and its application. The anti-fatigue composition employs a physical compounding technique using five raw materials: Haematococcus pluvialis, apple powder, fermented ginseng powder, spinach powder, and yerba mate extract. While this method improves saponin bioavailability to some extent through fermented ginseng powder, its core flaw lies in the fact that β-glucosidase secreted by lactic acid bacteria during fermentation actively hydrolyzes the C20 glycosidic bond of ginsenoside Rb1 to Rd, essentially abandoning the activity protection of the tetrasaccharide chain structure of ginsenoside Rb1. Furthermore, this method directly uses ordinary yerba mate extract without de-astringency treatment, failing to address the sensory defects caused by tannin astringency, and the use of a powder physical mixing process cannot meet the production requirements of liquid beverages.

[0004] Chinese patent application CN116035090A discloses an extraction process for tea beverages. Although this process uses immobilized tannin enzymes to treat green tea extract to remove bitterness, its carriers are chitosan microspheres and electrospun organic membranes. It targets the removal of ester-type catechins in green tea and lacks a hydrophobic substrate enrichment design that targets the tannin characteristics of yerba mate. More importantly, its final product does not involve ginsenoside active ingredients, and therefore completely fails to consider the impact of de-bitterness removal process conditions on the stability of ginsenoside Rb1, and does not propose a technical concept for protecting ginsenoside Rb1 in the same system.

[0005] In summary, existing technologies have consistently failed to achieve the dual objectives of efficient deastringency removal and low-loss protection of ginsenosides in Paraguayan tea within the same reaction system. Therefore, there is an urgent need to develop a preparation method that can synergistically resolve the aforementioned technical contradictions. Summary of the Invention

[0006] To solve the above-mentioned technical problems, the present invention provides a method for preparing a composition for relieving physical fatigue, comprising the following steps: Preparation of S1 ginseng extract: Ginseng raw material was extracted at 25-35℃ for 3-5 hours, and ginseng extract was obtained by membrane separation and purification.

[0007] Preparation of S2 Immobilized Enzyme Preparation: The enzyme source is adsorbed and immobilized on a mesoporous silica support modified with C8-C18 alkyl hydrophobicity to obtain the immobilized enzyme preparation. Immobilizing the enzyme source on the support solves the problem of easy inactivation and difficulty in separation from the product of traditional free enzymes under acidic deastringency conditions. The immobilized enzyme preparation can directionally remove tannins and astringent substances from Paraguayan tea while retaining effective components such as caffeine and total polyphenols. Taking advantage of the hydrophobic nature of tannin molecules in Paraguayan tea, C8-C18 alkylsilane chains are used to modify the surface of mesoporous silica. This allows for the pre-enrichment of tannin substrates at the pore inlet of the support through hydrophobic interactions, which can greatly improve the catalytic efficiency of tannin enzymes.

[0008] S3 deastringency reaction: Paraguayan tea extract is mixed with immobilized enzyme preparation, nitrogen is purged to remove oxygen, and the temperature is raised to 40-50℃. The pH value is adjusted to 5.0-6.0 using citrate-disodium hydrogen phosphate buffer, and the reaction is carried out for 1.5-2.5 hours. Under these conditions, tanninase can exhibit high catalytic activity. At this time, ginseng extract is not added to the system, which can avoid the risk of degradation of ginsenoside Rb1.

[0009] S4 Protective Reaction: Adjust the pH of the reaction system to 6.5-7.0, cool to 30-35℃, and then add ginseng extract to the reaction system at a mass ratio of 20-40:1. React for 0.5-1.0 hours. By adjusting the pH back to near neutral, not only is the activity of residual tannins reduced, but more importantly, the activity of H+ is eliminated. + The electrophilic attack driving force on the glycosidic bond of ginsenoside Rb1, combined with a low-temperature environment, can achieve "in-situ protection" of the active ingredient.

[0010] S5 enzyme recovery and concentration: Immobilized enzyme preparations were separated and recovered through a ceramic membrane filtration system with a pore size of 0.22-0.45μm. The filtrate was collected and concentrated under vacuum at 30-35℃ to obtain a concentrated extract of de-astringent Paraguayan tea and ginseng. S6 Final Product Blending and Homogenization: Mix 35-55 parts by weight of the de-astringent Paraguayan tea and ginseng extract concentrate with 5-12 parts by weight of the compound nutritional fortifier, 3-7 parts by weight of the natural sweetener, and 40-60 parts by weight of the deionized water. Homogenize at 15-20 MPa for 10-15 minutes, then sterilize and fill to obtain the finished product. The compound nutritional fortifier is composed of 2-5 parts by weight of B vitamins, 2-4 parts by weight of taurine, and 1-3 parts by weight of electrolyte salts. The natural sweetener is composed of erythritol and mogroside in a mass ratio of 2:1 to 6:1.

[0011] The core of this preparation method lies in the design of the sequence and conditions for steps S3→S4→S5: First, step S3 is carried out at pH 5.0-6.0 and 40-50℃, which are the optimal pH and temperature for tannin enzymes, enabling efficient hydrolysis of astringent tannins in Paraguayan tea extract. At this stage, ginseng extract has not yet been added to the system, thus avoiding potential hydrolytic damage to ginsenoside Rb1 under acidic conditions.

[0012] Secondly, in step S4, the pH of the system is adjusted back to near neutral 6.5-7.0 by adding phosphate buffer and the temperature is simultaneously lowered to 30-35℃. Then, ginseng extract is introduced. This not only significantly reduces the residual trace tannin enzyme activity, but also the neutral environment is conducive to inhibiting the hydrolysis of saponin glycosidic bonds, thereby achieving high retention of ginsenoside Rb1. Finally, step S5 involves rapid recovery of the immobilized enzyme via membrane filtration and vacuum concentration at low temperature to avoid thermal degradation of ginsenoside Rb1.

[0013] By implementing the above-described process steps in a sequential manner within the same system, continuous operation can be achieved, thus realizing the dual goals of efficient deastringency removal and high retention of ginsenoside Rb1. This integrated design not only significantly shortens the process flow but also effectively avoids the risk of cross-contamination that is easily introduced by traditional two-stage processes, thereby significantly improving the stability of active ingredients such as ginsenoside Rb1 and caffeine.

[0014] The finished product obtained through the above process exhibits a significantly improved rough and bitter taste. The synergistic effect of ginsenoside Rb1 and caffeine from Paraguayan tea in the formula exerts anti-fatigue effects through multiple pathways, including regulating the central nervous system, enhancing physical endurance, and boosting basal metabolism. The compound nutritional fortifier provides energy metabolism support and electrolyte balance, while the natural sweetener provides a pure sweetness, avoiding drastic fluctuations in blood sugar. All components work synergistically to create a functional beverage with excellent sensory quality and anti-fatigue function.

[0015] Preferably, the enzyme source is tanninase with an enzyme activity ≥500U / g, to ensure the catalytic efficiency of the immobilized enzyme per unit mass, thereby reducing the amount of enzyme preparation used while ensuring the deastringency effect.

[0016] Preferably, the mesoporous silica has a pore size range of 10-20 nm, a particle size of 1-10 μm, and a specific surface area greater than 200 m². 2 / g. The pore size of 10-20nm is sufficient to accommodate tanninase molecules and allow substrate to diffuse freely, while avoiding enzyme leakage due to excessively large pores; the particle size of 1-10μm facilitates subsequent filtration and recovery through ceramic membranes; the high specific surface area provides sufficient enzyme immobilization sites, increases the enzyme loading per unit volume of carrier, thereby shortening the deastringency reaction time and reducing the exposure time of ginsenoside Rb1 in acidic buffer.

[0017] Preferably, in step S3, the solids content of the Paraguayan tea extract is 8-12%, and the amount of immobilized enzyme preparation added is 0.5-1.5% of the substrate mass. By controlling the solids content of the extract and the amount of enzyme added, it is ensured that the substrate concentration and enzyme amount are within the optimal reaction kinetic range: if the solids content is too low, the production efficiency will decrease; if it is too high, it may inhibit enzyme activity or increase the viscosity of the system, affecting mass transfer. The 0.5-1.5% immobilized enzyme preparation addition can achieve the target of ≥72.5% removal rate of tannin-like astringent substances within 1.5-2.5 hours after optimization, avoiding excessively prolonging the reaction time or additional enzyme costs.

[0018] Preferably, in step S4, the pH of the reaction system is adjusted to 6.5-7.0 by adding a 0.01-0.05 mol / L disodium hydrogen phosphate-sodium dihydrogen phosphate buffer solution. This buffer system has excellent buffering capacity within the pH range of 6.5-7.0, can stably maintain a neutral environment, and can effectively inhibit the acid-catalyzed hydrolysis of ginsenoside Rb1 to ginsenoside Rd; at the same time, the components of this buffer solution are safe and non-toxic, with low ionic strength, and have no adverse effects on subsequent processes or the taste of the final product.

[0019] Preferably, in step S5, the vacuum level during vacuum concentration is maintained at -0.08 to -0.09 MPa, and the concentration is carried out until the solid content is 35-45%. This condition can effectively lower the boiling point, prevent the degradation of heat-sensitive components, and ensure the fluidity of the liquid.

[0020] Preferably, in step S6, the sterilization method is ultra-high temperature (UHT) instantaneous sterilization at a temperature of 135-140°C for 3-5 seconds. UHT instantaneous sterilization achieves commercial sterility requirements in a very short time. Compared with traditional pasteurization or long-term high-temperature sterilization, it can significantly shorten the exposure time of heat-sensitive components while preserving the unique aroma components of Paraguayan tea, ensuring that the final product has both excellent anti-fatigue activity and good sensory quality.

[0021] Another objective of this application is to provide a composition for relieving physical fatigue, which is prepared by the above-described preparation method.

[0022] The beneficial effects are as follows: This application utilizes immobilized enzyme preparations to catalyze the deastringency removal of Paraguayan tea extract, combined with a stepwise temperature and pH control strategy. The deastringency removal reaction and ginsenoside protection reaction are completed sequentially within the same reaction system, achieving a tannin removal rate of over 72.5% and a caffeine retention rate of over 86.9%. Simultaneously, it effectively inhibits the hydrolysis of ginsenoside Rb1, achieving an Rb1 retention rate of over 82.5%, significantly improving the anti-fatigue activity and sensory acceptability of the final product. Compared to existing technologies, this invention, through continuous operation within the same system, not only eliminates the astringency defect of yerba mate but also locks in the core active ingredients of ginseng through precise process control. The process is simpler and more efficient, with a lower probability of cross-contamination, and the product's effective components are more stable. Detailed Implementation

[0023] The present invention will now be described in further detail with reference to specific embodiments, so that those skilled in the art can more clearly understand the present invention.

[0024] The following embodiments are for illustrative purposes only and are not intended to limit the scope of the invention. All other embodiments obtained by those skilled in the art based on the specific embodiments of the invention without inventive effort are within the protection scope of the invention. The technical solutions of the various embodiments can be combined with each other, but this must be based on the ability of those skilled in the art to implement them. When the combination of technical solutions is contradictory or impossible to implement, it should be considered that such a combination of technical solutions does not exist and is not within the protection scope claimed by the invention.

[0025] Enzyme activity, also known as enzyme activity, refers to the ability of an enzyme to catalyze a specific chemical reaction. Its magnitude can be characterized by the conversion rate of the chemical reaction; the faster the rate, the higher the activity. The unit of enzyme activity is defined as the amount of enzyme that converts 1 micromolar of substrate per minute under optimal conditions, which is 1 unit (U).

[0026] In the embodiments of this invention, unless otherwise specified, all raw material components are commercially available products well-known to those skilled in the art; in the embodiments of this invention, unless specifically indicated, the technical means used are all conventional means well-known to those skilled in the art. Where specific experimental steps or conditions are not specified in the embodiments, they can be performed according to the conventional experimental steps or conditions described in the literature in this field. Reagents or instruments whose manufacturers are not specified are all conventional reagent products that can be obtained commercially.

[0027] Raw material source: The ginseng raw material is 40-mesh ginseng powder, which is made from whole sun-dried ginseng roots produced in Changbai Mountain, Jilin Province, with a specification of (10±0.5) g / root. Tanninase, provided by Beijing Weinuoen Biotechnology Co., Ltd., with enzyme activity ≥500U / g; Mesoporous silica with pore size controlled at 10-20 nm, particle size of 1-10 μm, and specific surface area >200 m². 2 / g, purchased from Zhongke Leiming (Beijing) Technology Co., Ltd.; All other raw and auxiliary materials are commercially available.

[0028] Example 1 A method for preparing a composition for relieving physical fatigue includes the following steps: Preparation of S1 ginseng extract: Ginseng raw material, namely 40-mesh ginseng powder, was added to 65% (v / v) ethanol aqueous solution at a material-to-liquid ratio of 1:10 (g / mL), and extracted at 25℃ for 3 hours. After filtration, the filtrate was concentrated under reduced pressure at 45℃ until no alcohol odor was detected. The filtrate was purified by ultrafiltration membrane (molecular weight cutoff 1 kDa) and then freeze-dried under vacuum to obtain ginseng extract.

[0029] Preparation of S2 immobilized enzyme preparation: Mesoporous silica (pore size range of 10-20 nm, particle size of 1-10 μm, specific surface area greater than 200 m²) was used. 2 Mesoporous silica (C8) was dispersed in anhydrous toluene at a mass-to-volume ratio of 1:10 (g / mL) and ultrasonically dispersed for 20 min. Under nitrogen protection, 15% (by mass) of n-octyltrimethoxysilane coupling agent was added, and the mixture was heated to 80 °C and refluxed for 12 h. After the reaction, the mixture was cooled to 20 °C, centrifuged to collect the solid, washed three times each with anhydrous ethanol and acetone, and vacuum dried at 60 °C for 6 h to obtain a C8 alkyl hydrophobically modified mesoporous silica support. The C8 alkyl hydrophobically modified mesoporous silica support was suspended in 0.05 mol / L citrate-disodium hydrogen phosphate buffer at pH 5.0 at a mass-to-volume ratio of 1:8 (g / mL) and ultrasonically dispersed for 15 min. A tanninase with an activity ≥500 U / g was added at a mass ratio of 1:3 (enzyme source to support). The mixture was shaken and adsorbed at 4 °C and 100 rpm for 12 h. After adsorption, the mixture was subjected to pH 5.0. Wash three times with 0.05 mol / L citrate-disodium hydrogen phosphate buffer to remove unadsorbed free enzyme, then freeze-dry under vacuum to obtain the immobilized enzyme preparation.

[0030] S3 Deastringency Reaction: Paraguayan tea extract with a solid content of 8% was mixed with an immobilized enzyme preparation. The amount of immobilized enzyme preparation added was 0.5% of the mass of the Paraguayan tea extract substrate. Nitrogen was purged for 20-30 min to remove oxygen, the temperature was raised to 40℃, and the pH was adjusted to 5.0 using 0.01 mol / L citrate-disodium hydrogen phosphate buffer. The reaction was carried out for 1.5 h.

[0031] S4 protection reaction: Add 0.01 mol / L disodium hydrogen phosphate-sodium dihydrogen phosphate buffer to the reaction system to adjust the pH of the reaction system to 6.5, cool to 30℃, and then add ginseng extract to the reaction system at a mass ratio of 20:1, and react for 0.5 h.

[0032] S5 Enzyme Recovery and Concentration: Immobilized enzyme preparations were separated and recovered using a ceramic membrane filtration system with a pore size of 0.22 μm. The filtrate was collected and concentrated under vacuum at 30 °C and -0.08 MPa to a solid content of 35%, yielding a concentrated extract of de-astringent Paraguayan tea and ginseng.

[0033] S6 Final Product Blending and Homogenization: According to the mass ratio, 35 parts of the above-mentioned de-astringent Paraguayan tea and ginseng extract concentrate, 5 parts of compound nutritional fortifier, 3 parts of natural sweetener, and 40 parts of deionized water are mixed and homogenized at 15 MPa pressure for 10 minutes. The mixture is then filled and sterilized using ultra-high temperature instantaneous sterilization (135℃, 5 seconds) to obtain the finished product. The compound nutritional fortifier is composed of 2 parts of vitamin B complex, 2 parts of taurine, and 1 part of electrolyte salt according to the mass ratio. The vitamin B complex is composed of 2 parts of vitamin B1 and 2 parts of vitamin B2. The natural sweetener is composed of erythritol and mogroside in a mass ratio of 2:1.

[0034] Example 2 A method for preparing a composition for relieving physical fatigue includes the following steps: Preparation of S1 ginseng extract: Ginseng raw material, namely 40-mesh ginseng powder, was added to 75% (v / v) ethanol aqueous solution at a material-to-liquid ratio of 1:15 (g / mL), and extracted at 30℃ for 4 hours. After filtration, the filtrate was concentrated under reduced pressure at 45℃ until no alcohol odor was detected. The filtrate was purified by ultrafiltration membrane (molecular weight cutoff 2kDa) and then freeze-dried under vacuum to obtain ginseng extract.

[0035] Preparation of S2 immobilized enzyme preparation: Mesoporous silica (pore size range of 10-20 nm, particle size of 1-10 μm, specific surface area greater than 200 m²) was used. 2Mesoporous silica (C12) was dispersed in anhydrous toluene at a mass-to-volume ratio of 1:15 (g / mL) and ultrasonically dispersed for 25 min. Under nitrogen protection, 25% (by mass) of dodecyltrimethoxysilane coupling agent was added, and the mixture was refluxed at 100 °C for 18 h. After the reaction, the mixture was cooled to 25 °C, centrifuged to collect the solid, washed four times each with anhydrous ethanol and acetone, and vacuum dried at 70 °C for 9 h to obtain a C12 alkyl hydrophobically modified mesoporous silica support. The C12 alkyl hydrophobically modified mesoporous silica support was suspended in a 0.07 mol / L citrate-disodium hydrogen phosphate buffer solution at pH 5.5 at a mass-to-volume ratio of 1:10 (g / mL) and ultrasonically dispersed for 17 min. A tanninase with an activity ≥500 U / g was added at a mass ratio of 1:5 (enzyme source to support). The mixture was shaken and adsorbed at 8 °C and 120 rpm for 18 h. After adsorption, the mixture was subjected to pH... The enzyme was washed four times with 0.07 mol / L citrate-disodium hydrogen phosphate buffer at 5.5°C to remove unadsorbed free enzyme, and then freeze-dried under vacuum to obtain the immobilized enzyme preparation.

[0036] S3 Deastringency Reaction: Paraguayan tea extract with a solid content of 10% was mixed with an immobilized enzyme preparation. The amount of immobilized enzyme preparation added was 1.0% of the mass of the Paraguayan tea extract substrate. Nitrogen was purged for 25 min to remove oxygen, the temperature was raised to 45℃, and the pH was adjusted to 5.5 using 0.03 mol / L citrate-disodium hydrogen phosphate buffer. The reaction was carried out for 2 h.

[0037] S4 protection reaction: Add 0.03 mol / L disodium hydrogen phosphate-sodium dihydrogen phosphate buffer to the reaction system to adjust the pH of the reaction system to 7.0, cool to 32℃, and then add ginseng extract to the reaction system at a mass ratio of 30:1, and react for 0.7 h.

[0038] S5 Enzyme Recovery and Concentration: Immobilized enzyme preparations were separated and recovered using a ceramic membrane filtration system with a pore size of 0.35 μm. The filtrate was collected and vacuum concentrated to a solid content of 40% at 32℃ and -0.08 MPa to obtain a concentrated extract of de-astringent Paraguayan tea and ginseng.

[0039] S6 Final Product Blending and Homogenization: According to the mass ratio, 45 parts of the above-mentioned de-astringent Paraguayan tea and ginseng extract concentrate are mixed with 8 parts of compound nutritional fortifier, 5 parts of natural sweetener and 50 parts of deionized water, homogenized at 17MPa pressure for 12 minutes, filled and sterilized by ultra-high temperature instantaneous sterilization (140℃, 3 seconds) to obtain the finished product; the compound nutritional fortifier is composed of 4 parts of vitamin B complex, 3 parts of taurine and 2 parts of electrolyte salt according to the mass ratio; the vitamin B complex is composed of 2 parts of vitamin B1 and 2 parts of vitamin B6; the natural sweetener is composed of erythritol and mogroside in a mass ratio of 4:1.

[0040] Example 3 A method for preparing a composition for relieving physical fatigue includes the following steps: Preparation of S1 ginseng extract: Ginseng raw material, namely 40-mesh ginseng powder, was added to 85% (v / v) ethanol aqueous solution at a material-to-liquid ratio of 1:20 (g / mL), and extracted at 35℃ for 5 hours. After filtration, the filtrate was concentrated under reduced pressure at 45℃ until no alcohol odor was detected. The filtrate was purified by ultrafiltration membrane (molecular weight cutoff 3kDa) and then freeze-dried under vacuum to obtain ginseng extract.

[0041] Preparation of S2 immobilized enzyme preparation: Mesoporous silica (pore size range of 10-20 nm, particle size of 1-10 μm, specific surface area greater than 200 m²) was used. 2 Mesoporous silica (C18) was dispersed in anhydrous toluene at a mass-to-volume ratio of 1:20 (g / mL) and ultrasonically dispersed for 30 min. Under nitrogen protection, 35% of the mass of mesoporous silica was added as a coupling agent, and the mixture was heated to 110 °C and refluxed for 24 h. After the reaction, the mixture was cooled to 30 °C, centrifuged to collect the solid, washed five times each with anhydrous ethanol and acetone, and vacuum dried at 80 °C for 12 h to obtain a C18 alkyl hydrophobic modified mesoporous silica support. The C18 alkyl hydrophobic modified mesoporous silica support was suspended in 0.1 mol / L citrate-disodium hydrogen phosphate buffer at pH 6.0 at a mass-to-volume ratio of 1:15 (g / mL) and ultrasonically dispersed for 20 min. A tanninase with an activity ≥500 U / g was added at a mass ratio of 1:8. The mixture was shaken and adsorbed at 10 °C and 150 rpm for 24 h. After adsorption, the mixture was subjected to pH 6.0. The enzyme was washed five times with 0.1 mol / L citrate-disodium hydrogen phosphate buffer to remove unadsorbed free enzyme, and then freeze-dried under vacuum to obtain the immobilized enzyme preparation.

[0042] S3 Deastringency Reaction: Paraguayan tea extract with a solid content of 12% was mixed with an immobilized enzyme preparation. The amount of immobilized enzyme preparation added was 1.5% of the mass of the Paraguayan tea extract substrate. Nitrogen was purged for 30 min to remove oxygen, the temperature was raised to 50℃, and the pH was adjusted to 6.0 using 0.05 mol / L citrate-disodium hydrogen phosphate buffer. The reaction was carried out for 2.5 h.

[0043] S4 protection reaction: Add 0.05 mol / L disodium hydrogen phosphate-sodium dihydrogen phosphate buffer to the reaction system to adjust the pH of the reaction system to 7.0, cool to 35℃, and then add ginseng extract to the reaction system at a mass ratio of 40:1, and react for 1.0 h.

[0044] S5 Enzyme Recovery and Concentration: Immobilized enzyme preparations were separated and recovered using a ceramic membrane filtration system with a pore size of 0.45 μm. The filtrate was collected and vacuum concentrated to a solid content of 45% at 35℃ and -0.09 MPa to obtain a concentrated extract of de-astringent Paraguayan tea and ginseng.

[0045] S6 Final Product Blending and Homogenization: According to the mass ratio, 55 parts of the above-mentioned de-astringent Paraguayan tea and ginseng extract concentrate are mixed with 12 parts of compound nutrient fortifier, 7 parts of natural sweetener and 60 parts of deionized water, homogenized at 20MPa pressure for 15min, filled and sterilized by ultra-high temperature instantaneous sterilization (135℃, 3 seconds) to obtain the finished product; the compound nutrient fortifier is composed of 5 parts of vitamin B complex, 4 parts of taurine and 3 parts of electrolyte salt according to the mass ratio; the vitamin B complex is composed of 2 parts of vitamin B2 and 2 parts of vitamin B6; the natural sweetener is composed of erythritol and mogroside in a mass ratio of 6:1.

[0046] Comparative Example 1 The difference between this comparative example and Example 2 is that step S4 (low-temperature, low-activity mixing protection reaction) is omitted. That is, after the de-astringency step in S3, pH adjustment and cooling are not performed, and ginseng extract is not added for a protection reaction. Instead, the immobilized enzyme recovery and vacuum concentration steps in S5 are directly executed. In the final product blending in S6, the ginseng extract is directly mixed with the de-astringent Paraguayan tea concentrate, compound nutrient fortifier, and other components using a simple physical mixing process. All other components, process steps, and parameters are the same as in Example 2.

[0047] Comparative Example 2 The difference between this comparative example and Example 2 is that the pH adjustment operation in step S4 is omitted. That is, after completing the deastringency step S3, no disodium hydrogen phosphate-sodium dihydrogen phosphate buffer is added, so that the pH value of the reaction system is maintained at pH 5.5 at the end of step S3. The temperature is directly lowered to 32°C, and then ginseng extract is added and reacted for 0.7 h. The remaining components, process steps, and parameters are the same as in Example 2.

[0048] Comparative Example 3 The difference between this comparative example and Example 2 is that the cooling process is not performed in step S4. That is, after completing the deastringency step S3 and adjusting the pH to 7.0, the system temperature is maintained at 45°C before adding the ginseng extract for reaction. The remaining components, process steps, and parameters are the same as in Example 2.

[0049] Comparative Example 4 The difference between this comparative example and Example 2 is that steps S4 and S5 are reversed. Specifically, the immobilized enzyme recovery step (S5) is performed first, followed by the low-temperature, low-activity mixed protective reaction (S4). After completing the deastringency step (S3), the immobilized enzyme is first separated and recovered using a 0.35 μm ceramic membrane filtration system, and the filtrate is collected. Then, a 0.03 mol / L disodium hydrogen phosphate-sodium dihydrogen phosphate buffer solution is added to the filtrate to adjust the pH to 7.0, the temperature is lowered to 32°C, and ginseng extract is added at a mass ratio of 30:1. The reaction is carried out for 0.7 h. Subsequent steps, such as vacuum concentration, are then performed. All other components, process steps, and parameters are the same as in Example 2.

[0050] Comparative Example 5 The difference between this comparative example and Example 2 is that the mesoporous silica support used in step S2 was not modified with C8-C18 alkyl hydrophobication. Specifically, the silane coupling agent modification step was omitted, and the original mesoporous silica (pore size 10-20 nm, particle size 1-10 μm, specific surface area >200 m²) was used directly. 2 The tanninase was adsorbed and immobilized using the same method to obtain the immobilized enzyme preparation. All other components, process steps, and parameters were the same as in Example 2.

[0051] Comparative Example 6 The difference between this comparative example and Example 2 is that, in step S2, when performing surface hydrophobic modification on the mesoporous silica support, an equal mass fraction of n-butyltrimethoxysilane coupling agent was used instead of n-dodecyltrimethoxysilane coupling agent. All other components, process steps, and parameters are the same as in Example 2.

[0052] Experimental Example 1: Determination of Retention Rate of Ginsenoside Rb1 and Tannin Removal Rate Experimental subjects: Samples from each embodiment and comparative example; Experimental methods: Determination of ginsenoside Rb1 content: HPLC method, C18 column (250 mm × 4.6 mm, 5 μm), mobile phase acetonitrile-water gradient elution (0-30 min, acetonitrile 20%-40%), flow rate 1.0 mL / min, detection wavelength 203 nm. A standard curve was prepared using ginsenoside Rb1 standard (purity ≥ 98%). Ginsenoside Rb1 retention rate = (measured ginsenoside Rb1 content in the final product / theoretical feed amount) × 100%.

[0053] Determination of ginsenoside Rd content: Under the same HPLC conditions as described above, qualitative and quantitative analysis was performed using ginsenoside Rd standard, and the accumulation of Rb1 hydrolysis products was monitored.

[0054] Tannin content determination: The total tannin content was determined using the Folin-Denis method with gallic acid as the standard. Tannin removal rate = [(initial content - residual content) / initial content] × 100%.

[0055] Caffeine content determination: HPLC method, C18 column, mobile phase methanol-water (30:70), detection wavelength 273nm.

[0056] Table 1. Test results of ginsenoside Rb1 retention rate and tannin removal rate. Note: Since ginsenoside Rb1 can generate other degradation products, the sum of the retention rate of ginsenoside Rb1 and the formation rate of ginsenoside Rd is not equal to 100%. Other degradation products are not the focus of this application, so they were not further quantitatively detected, and their experimental data are not shown in the table.

[0057] The optimal reaction conditions for tanninase (pH 5.0-6.0, 40-50℃) fall precisely within the sensitive degradation range of ginsenoside Rb1. The tetrasaccharide chain structure at the C20 position of ginsenoside Rb1 is readily hydrolyzed to Rd under acidic or heated conditions, leading to a significant decrease in its anti-fatigue activity. To address this technical contradiction, this invention employs a step-by-step strategy of "de-astringency followed by protection": Step S3 involves efficient de-astringency in a system devoid of ginsenoside Rb1, avoiding hydrolysis by acidic high temperatures; Step S4 achieves "active protection" through pH adjustment and synergistic cooling, raising the system pH from 5.0-6.0 to 6.5-7.0, utilizing the neutral environment to inhibit H+ degradation. + Electrophilic attack on the glycosidic bond of ginsenoside Rb1 inhibits acid-catalyzed hydrolysis of ginsenoside Rb1. At the same time, lowering the temperature to 30-35℃ significantly reduces residual enzyme activity and slows down the rate of non-enzymatic hydrolysis.

[0058] Table 1 shows that the retention rate of ginsenoside Rb1 in Examples 1-3 is significantly better than that in all comparative examples. At the same time, the tannin removal rate is also over 72%, and the caffeine retention rate is over 86%, indicating that the present invention can effectively lock in the core active ingredients while achieving efficient deastringency.

[0059] Comparative Example 1 aims to simulate the common process route of "first deastringent treatment, then simple compounding" in existing technologies. Due to the omission of the S4 protection step, the system after deastringent treatment is in the optimal environment of tannin enzyme at 45°C and pH 5.5 for a long time. Under these conditions, the residual enzyme activity continues to act, and the concentration process further aggravates the degradation of ginsenoside Rb1. Therefore, the retention rate of ginsenoside Rb1 is only 70.5%, and the formation rate of ginsenoside Rd is as high as 22.2%.

[0060] Although Comparative Example 2 was cooled, the pH was not adjusted. The acidic environment of pH 5.5 could still drive the acid-catalyzed hydrolysis of ginsenoside Rb1, so the retention rate of ginsenoside Rb1 was only 72.5%.

[0061] Although the pH of Comparative Example 3 was adjusted to 7.0, the temperature was not lowered simultaneously. The high temperature of 45°C may still activate a small number of non-inactivated tanninases on the immobilized enzyme, or accelerate other thermo-induced degradation pathways (such as oxidation and isomerization), resulting in a ginsenoside Rb1 retention rate of 73.1%, which is still significantly lower than that of the Example.

[0062] Comparative Example 4 reversed the order of steps S4 and S5, i.e., the enzyme preparation was recovered immediately after deastringency, and then the pH was adjusted before adding ginseng extract. Although the tannin removal rate was comparable to that of the example, the retention rate of ginsenoside Rb1 decreased significantly. This may be due to two reasons. Firstly, the immobilized enzyme carrier lost its ability to continuously adsorb and degrade trace amounts of tannins, leading to hydrophobic aggregation or even co-precipitation of residual tannins with ginsenosides during subsequent concentration, accelerating the non-enzymatic degradation of ginsenoside Rb1. Secondly, the process of filtering before adjusting pH changed the production from a single-stage continuous operation to a two-stage operation, resulting in operational contamination and affecting the stability of ginsenoside Rb1 and caffeine. Following the process steps of the example, adding ginsenoside Rb1 in step S4, in a neutral, low-temperature system with an immobilized enzyme carrier, may be more conducive to the formation of a stable supramolecular complex between ginsenoside Rb1 and the deastringent Paraguayan tea polyphenols. This complex protects ginsenoside Rb1 from acid-catalyzed hydrolysis through a steric shielding effect, thereby improving the retention rate of ginsenoside Rb1.

[0063] Comparative Example 5 used a carrier without hydrophobic modification. Due to the lack of enrichment effect on hydrophobic tannin molecules, the catalytic efficiency of the immobilized enzyme decreased significantly, and the tannin removal rate was only 63.5%. If the same level of deastringency is to be achieved, the reaction time must be extended or the amount of enzyme used must be increased, which will indirectly aggravate the degradation risk of ginsenoside Rb1 in subsequent process steps.

[0064] In Comparative Example 6, n-butyltrimethoxysilane coupling agent was used to replace n-dodecyltrimethoxysilane coupling agent in step S2 to modify the surface of the mesoporous silica support for hydrophobic modification. Due to the excessively short alkyl chain length, the hydrophobic interaction of the enzyme preparation was poor, and the substrate tannic acid could not be effectively enriched, resulting in a significant decrease in the catalytic efficiency of tanninase. Furthermore, due to the poor deastringency effect, the residual tannic acid increased, which was prone to hydrophobic aggregation or even co-precipitation with ginsenosides during the subsequent concentration process.

[0065] Experimental Example 2: Anti-fatigue animal experiment Experimental subjects: Samples from each embodiment and comparative example. Experimental methods: Two hundred male Kunming mice weighing 25-35g were selected and, after 7 days of acclimatization, were randomly divided into 10 groups of 20 mice each, with no significant differences among the groups. The mice were divided into a treatment group and a control group. The treatment group was administered 0.5 mL / 10g bw of the samples provided in the examples and comparative examples by gavage at 10:00 AM daily. The control group was administered an equal volume of physiological saline by gavage. Mice had free access to water during the gavage period. Exercise endurance and biochemical indicators were measured after 30 consecutive days of treatment.

[0066] (1) Swimming experiment On day 27 after gavage, mice were acclimatized to swimming. One hour after gavage, the mice were placed in water at 25±1℃ and swam for 10 minutes without weight-bearing. This acclimatization period lasted for 3 days, with the swimming time increasing by 5 minutes each day thereafter. On day 30, one hour after gavage, a swimming exhaustion test was conducted. Mice were fasted for 12 hours before swimming and had a lead weight of 6% of their body weight attached to their tails. The swimming exhaustion time for each group was recorded. The standard was that the mouse remained submerged for 8 seconds and was unable to surface for air. The swimming exhaustion time data for each group are shown in Table 2.

[0067] (2) Blood lactate content Blood lactate is a product of anaerobic metabolism during exercise, and its concentration is closely related to exercise intensity and fatigue level. When exercise intensity exceeds the aerobic metabolic threshold, lactate accumulates in muscles, leading to a decrease in pH and subsequently causing fatigue. Therefore, lactate is an important indicator of exercise-induced fatigue. Immediately after swimming, the tail tips of mice were disinfected with alcohol swabs, and blood was collected by tail cutting. A portable blood lactate analyzer was used to measure the lactate content of each group at various time points. The specific results are shown in Table 2.

[0068] Table 2. Test results of exhaustion time and blood lactate content for each example and comparative sample (n=20). ±s) Note: Data are presented as mean ± standard deviation (%) ±s) represents the mean, n=20 (number of independent experiments). Intergroup comparisons were performed using one-way ANOVA followed by Tukey's multiple comparison test. Compared with the blank control group, * indicates P<0.05, ** indicates P<0.01; compared with Example 2, # This indicates that P < 0.05. ## This indicates that P < 0.01.

[0069] Table 2 shows that the exhaustion swimming time and post-exercise blood lactate level in mice of each sample treatment group were significantly better than those of the blank control group, indicating that each composition has a certain anti-fatigue effect. Among them, the exhaustion time of Example 2 was significantly longer than that of each comparative group, and the post-exercise blood lactate level was significantly lower than that of each comparative group, indicating that the anti-fatigue effect of Example 2 was significantly better than that of each comparative group. This is highly consistent with the trend of ginsenoside Rb1 retention rate in Table 1, that is, the higher the ginsenoside Rb1 retention rate, the better the anti-fatigue effect.

[0070] Experimental Example 3: Sensory Evaluation Experimental subjects: Each embodiment and comparative sample.

[0071] Experimental methods: Twenty trained sensory evaluators (half male and half female, aged 22-35) scored the samples on astringency intensity and overall acceptability (0-10 points, lower astringency score better, higher acceptability score better). Appropriate amounts of samples were taken, randomly numbered, and provided to the evaluators in a random order. Sensory evaluators did not consume foods with pungent tastes before evaluation, scored the randomly provided samples, and rinsed their mouths with purified water after each evaluation. The test results are shown in Table 3.

[0072] Table 3. Sensory evaluation results of each embodiment and comparative sample (n=20, ±s) The astringency in Paraguayan tea mainly originates from polyphenolic substances such as tannins. These substances bind to proline in saliva, causing precipitation or aggregation, leading to the loss of oral lubrication and a dry, rough, astringent sensation. Tanninase can specifically hydrolyze the ester and phenolic carboxyl bonds in tannins, producing gallic acid and glucose. These products do not have the ability to cross-link with salivary proteins to form precipitates, thus significantly reducing astringency. As shown in Table 3, Comparative Example 5, due to the lack of hydrophobic modification of the carrier, had the lowest tannin removal rate, resulting in an astringency score as high as 5.6 and the lowest acceptability. Comparative Example 6, due to the poor catalytic effect of the enzyme preparation, had a lower tannin removal rate, and its astringency score and acceptability were only slightly better than Comparative Example 5. Although the other comparative examples were similar to the examples in terms of astringency removal effect, their overall quality was inferior to the examples due to the low retention rate of ginsenoside Rb1 and poor anti-fatigue effect. Therefore, this invention can achieve high retention of active ingredients and enhanced functionality while ensuring a good taste.

[0073] The above are merely embodiments of the present invention and are not intended to limit the invention. Various modifications and variations can be made to the present invention by those skilled in the art. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principle of the present invention should be included within the scope of the claims of the present invention.

Claims

1. A method for preparing a composition for relieving physical fatigue, characterized in that, Includes the following steps: Preparation of S1 ginseng extract: Ginseng raw material was extracted at 25-35℃ for 3-5 hours, and the ginseng extract was obtained by membrane separation and purification; S2 Immobilized Enzyme Preparation: The enzyme source was adsorbed and immobilized on a mesoporous silica support modified with C8-C18 alkyl hydrophobicity to obtain the immobilized enzyme preparation. S3 Deastringency Reaction: Mix Paraguayan tea extract with the immobilized enzyme preparation, heat to 40-50℃, adjust pH to 5.0-6.0, and react for 1.5-2.5 hours; S4 Protective reaction: Adjust the pH of the reaction system to 6.5-7.0, cool to 30-35℃, add the ginseng extract to the reaction system at a mass ratio of 20-40:1, and react for 0.5-1.0 h; S5 enzyme recovery and concentration: Immobilized enzyme preparations were separated and recovered through a ceramic membrane filtration system with a pore size of 0.22-0.45μm. The filtrate was vacuum concentrated at 30-35℃ to obtain a concentrated extract of de-astringent Paraguayan tea and ginseng. S6 Final Product Preparation and Homogenization: Mix with acceptable excipients, homogenize, and sterilize to obtain the finished product.

2. The method for preparing the composition for relieving physical fatigue according to claim 1, characterized in that, In step S2, the enzyme source is tanninase with an enzyme activity ≥500 U / g.

3. The method for preparing the composition for relieving physical fatigue according to claim 1, characterized in that, In step S2, the mesoporous silica has a pore size range of 10-20 nm, a particle size of 1-10 μm, and a specific surface area greater than 200 m². 2 / g.

4. The method for preparing the composition for relieving physical fatigue according to claim 1, characterized in that, In step S3, the solid content of the Paraguayan tea extract is 8-12%, and the amount of the immobilized enzyme preparation added is 0.5-1.5% of the substrate mass.

5. The method for preparing the composition for relieving physical fatigue according to claim 1, characterized in that, In step S3, the pH value is adjusted to 5.0-6.0 using a citrate-disodium hydrogen phosphate buffer solution; in step S4, the pH value of the reaction system is adjusted to 6.5-7.0 using a disodium hydrogen phosphate-sodium dihydrogen phosphate buffer solution.

6. The method for preparing the composition for relieving physical fatigue according to claim 1, characterized in that, In step S5, the vacuum level is maintained at -0.08 to -0.09 MPa during vacuum concentration, and the concentration is carried out until the solid content is 35-45%.

7. The method for preparing the composition for relieving physical fatigue according to claim 1, characterized in that, In step S6, the acceptable excipients include a compound nutritional fortifier and a natural sweetener, wherein the compound nutritional fortifier is composed of a vitamin B complex, taurine, and electrolyte salts in parts by weight.

8. The method for preparing the composition for relieving physical fatigue according to claim 7, characterized in that, In step S6, the natural sweetener is a combination of erythritol and mogroside.

9. The method for preparing the composition for relieving physical fatigue according to claim 1, characterized in that, In step S6, homogenization needs to be performed at a pressure of 15-20 MPa for 10-15 minutes; sterilization is performed by ultra-high temperature instantaneous sterilization at a temperature of 135-140℃ for 3-5 seconds.

10. A composition for relieving physical fatigue, characterized in that, It is prepared by any one of the preparation methods described in claims 1-9.

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