A composition, its preparation and use

Abstract

The application discloses a composition comprising a complex microalgae active ingredient, taurine and vitamin C; wherein the complex microalgae active ingredient is composed of chlorella vulgaris CGF extract, spirulina phycocyanin and haematococcus pluvialis astaxanthin microcapsules, the complex microalgae active ingredient is compounded with taurine and vitamin C, and has an anti-fatigue homeostasis function.

Description

Technical Field

[0001] This application belongs to the field of functional beverage technology, specifically relating to a multi-target anti-fatigue homeostatic functional beverage with multiple compound microalgae active ingredients as the core, compounded with taurine and vitamin C, and its preparation method and application. Background Technology

[0002] With the accelerated pace of modern life and increased awareness of exercise and health, the market size of anti-fatigue functional beverages continues to grow. Traditional functional beverages mainly use taurine, caffeine, and synthetic vitamins as core active ingredients to achieve a short-term energizing effect through nerve stimulation and energy replenishment. However, they have drawbacks such as single target of action, heavy metabolic burden, and inability to fundamentally alleviate oxidative fatigue.

[0003] Microalgae, as an important source of natural bioactive substances, has become a cutting-edge international research area in sports nutrition. Multiple evidence-based medical studies have confirmed that Chlorella growth factor (CGF) can promote cell repair and enhance physical endurance; Spirulina platensis phycocyanin has significant antioxidant and lactic acid scavenging activity, accelerating post-exercise recovery; and natural astaxanthin from Haematococcus pluvialis, as a potent fat-soluble antioxidant, can penetrate cell membranes to protect mitochondrial function and reduce exercise-induced oxidative damage (PMC). Existing technologies include reports on the combination of astaxanthin, taurine, and vitamin C in sports drinks (CN112515070A), a patent related to Chlorella complex vitamin drinks (CN110859254A), and a patent related to complex microalgae powder (CN105746774A), but these technologies generally face the following technical bottlenecks:

[0004] 1. The selection of microalgae active ingredients is crude: algae powder or single extracts are often added directly without scientific compounding of multiple algae species to target anti-fatigue. As a result, the active ingredients have low purity, many impurities, and poor bioavailability.

[0005] 2. Incomplete antioxidant system: Relying solely on a single water-soluble or fat-soluble antioxidant cannot construct a water-oil dual-phase protection system, resulting in incomplete removal of free radicals generated at multiple sites during exercise.

[0006] 3. Poor activity stability: Phycocyanin is sensitive to heat, light and pH, and astaxanthin is easily oxidized and degraded; Vitamin C is easily oxidized in aqueous systems and cannot effectively protect fat-soluble active ingredients, resulting in severe efficacy degradation during shelf life.

[0007] 4. Unclear synergistic mechanism: The combination of microalgae components with taurine and vitamin C is mostly a simple additive, without optimization of the ratio based on the target of action, making it difficult to achieve a synergistic effect of 1+1>2.

[0008] 5. Poor flavor and palatability: Microalgae extracts generally have a characteristic algal odor, which existing products often rely on high sugar content and large amounts of flavoring to mask, which does not conform to the current trend of health-conscious consumption.

[0009] Therefore, developing a multi-target synergistic, stable, palatable, and fatigue-fighting functional beverage based on a microalgae combination, taurine, and vitamin C complex system is a technical problem that urgently needs to be solved in this field. Summary of the Invention

[0010] To address the aforementioned technical problems, this application provides a composition comprising a complex microalgae active ingredient, taurine, and vitamin C; wherein the complex microalgae active ingredient is composed of Chlorella CGF extract, Spirulina phycocyanin, and Haematococcus pluvialis astaxanthin microcapsules.

[0011] This application is achieved through the following technical solution:

[0012] The first aspect of this application provides a composition.

[0013] In some embodiments, a composition comprises a complex microalgae active ingredient, taurine, and vitamin C; wherein the complex microalgae active ingredient is composed of Chlorella CGF extract, Spirulina phycocyanin, and Haematococcus pluvialis astaxanthin microcapsules.

[0014] In some embodiments, the composite microalgae active ingredients are composed of 1-10 parts by weight of Chlorella CGF extract, 1-7 parts by weight of Spirulina phycocyanin, and 0.5-5 parts by weight of Haematococcus pluvialis astaxanthin microcapsules.

[0015] In some embodiments, the mass fraction of Chlorella CGF extract in the composite microalgae active ingredient can be 1 part, 2 parts, 3.0 parts, 4.0 parts, 5.0 parts, 6.0 parts, 7.0 parts, 8.0 parts, 9.0 parts, or 10.0 parts.

[0016] In some embodiments, the amount of spirulina phycocyanin protein in the composite microalgae active ingredient can be 1.0 part, 2.0 part, 3.0 part, 4.0 part, 5.0 part, 6.0 part, or 7.0 part.

[0017] In some embodiments, the mass fraction of Haematococcus pluvialis astaxanthin microcapsules in the composite microalgae active ingredient can be 0.5 parts, 1.0 parts, 1.5 parts, 2.0 parts, 2.5 parts, 3.0 parts, 3.5 parts, 4.0 parts, 4.5 parts, or 5.0 parts.

[0018] In some embodiments, the composite microalgae active ingredients are composed of 2-6 parts by weight of Chlorella CGF extract, 1-4 parts of Spirulina phycocyanin, and 1-3 parts of Haematococcus pluvialis astaxanthin microcapsules.

[0019] In some embodiments, the mass ratio of the Chlorella CGF extract, Spirulina phycocyanin, and Haematococcus pluvialis astaxanthin microcapsules is 2:1.5:1.

[0020] In some embodiments, the mass ratio of Chlorella CGF extract, Spirulina phycocyanin, and Haematococcus pluvialis astaxanthin microcapsules is 4:3:2.

[0021] In some embodiments, the mass ratio of Chlorella CGF extract, Spirulina phycocyanin, and Haematococcus pluvialis astaxanthin microcapsules is 2:1:1.

[0022] In some embodiments, the mass ratio of the Chlorella CGF extract, Spirulina phycocyanin, and Haematococcus pluvialis astaxanthin microcapsules is 1:1:1.

[0023] In some embodiments, the Chlorella is Chlorella proteoglycans.

[0024] In some embodiments, the spirulina is Spirulina platensis.

[0025] In some embodiments, the total nucleotide and amino acid content of the Chlorella vulgaris CGF extract is ≥35%. For example, the total content can be 35%, 36%, 37%, 38%, 39%, 40%, 41%, 42%, 43%, 44%, 45%, 46%, 47%, 48%, 49%, 59%, 51%, 52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%, 60%, 62%, 64%, 66%, 68%, or 70%.

[0026] In some embodiments, the purity of the phycocyanin from *Spirulina platensis* is ≥25%. The purity is the percentage by mass of phycocyanin in the dry spirulina powder. Examples of phycocyanin purity include 25%, 30%, 35%, 40%, 41%, 42%, 43%, 44%, 45%, 46%, 47%, 48%, 49%, 50%, 40%, 51%, 52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%, 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 95%, or 100%.

[0027] In some embodiments, the color valence E of the Spirulina platensis phycocyanin is... 18 ≥60.

[0028] In some embodiments, the astaxanthin content in the Haematococcus pluvialis astaxanthin microcapsules is ≥2.0% by mass. Examples of the mass content include 2.0%, 2.1%, 2.2%, 2.3%, 2.4%, 2.5%, 2.6%, 2.7%, 2.8%, 2.9%, 3.0%, 3.1%, 3.2%, 3.3%, 3.4%, 3.5%, 3.6%, 3.7%, 3.8%, 3.9%, or 4.0%.

[0029] To better realize the efficacy of this application, preferably, the Chlorella vulgaris CGF extract, Spirulina platensis phycocyanin, and Haematococcus pluvialis astaxanthin microcapsules are all prepared in-house.

[0030] In some embodiments, the Chlorella proteoglycans CGF extract is obtained by steam thermal swelling and cell wall disruption, hot water extraction, ultrafiltration, and concentration of Chlorella proteoglycans.

[0031] In some embodiments, the Chlorella vulgaris CGF extract is obtained by the following extraction method:

[0032] (1) Low temperature soaking: Mix Chlorella powder with water at a weight ratio of 1: (10-30) and stir evenly. Soak at a low temperature of 4-10℃ for 1-3 hours.

[0033] (2) Steam thermal swelling and cell wall breaking treatment: The soaked algal solution is subjected to cell wall breaking treatment by steam thermal swelling and cell wall breaking. The cell wall breaking temperature is 100-120℃, the pressure is 150-200Pa, and the treatment time is 60-120 minutes.

[0034] (3) Hot water extraction: After the cell wall is broken, hot water extraction is carried out at 35-55℃ and stirring speed of 100-200rpm for 60-180 minutes; preferably, deoxyribonuclease is added during the extraction reaction, and the amount added is 5 million to 10 million units / L of reaction solution;

[0035] (4) Centrifugation: The extract is centrifuged to remove insoluble matter, with a centrifugation speed >4500 rpm;

[0036] (5) Ultrafiltration treatment: The supernatant obtained by centrifugation is subjected to ultrafiltration treatment using an ultrafiltration membrane with a molecular weight cutoff of 1 to 50 kDa and a temperature of 25 to 40 °C;

[0037] (6) Concentration and drying: Collect the ultrafiltration concentrate and dry it to obtain Chlorella vulgaris CGF extract.

[0038] In some embodiments, the ultrafiltration method for extracting Chlorella vulgaris CGF extract employs a hollow fiber ultrafiltration system with a membrane pore size of 0.1 μm.

[0039] In some embodiments, the ultrafiltration membrane used in the extraction method of Chlorella vulgaris CGF extract has a molecular weight cutoff of 3 to 10 kDa, for example, 3 kDa, 4 kDa, 5 kDa, 6 kDa, 7 kDa, 8 kDa, 9 kDa or 10 kDa.

[0040] In some embodiments, the steam thermal swelling and cell wall disruption method for extracting Chlorella vulgaris CGF extract employs multiple streams of steam at different pressures and temperatures for synergistic thermal swelling and disruption. Specifically, one stream consists of 150 Pa and 100°C, another of 180 Pa and 110°C, and yet another of 200 Pa and 120°C, repeated three times, with a total processing time of 60–120 minutes. The synergistic effect of the temperature and pressure gradients achieves complete cell wall rupture, facilitating thorough extraction in the subsequent step.

[0041] In some embodiments, the preparation method of the Spirulina platensis phycocyanin includes Spirulina platensis powder extraction, centrifugation of the extract, purification of the centrifugation supernatant, and drying.

[0042] In some embodiments, the extraction of *Spirulina platensis* powder in the preparation method of *Spirulina platensis* phycocyanin includes a swelling method, a freeze-thaw method, or a high-speed homogenization method. In some specific embodiments, the swelling method involves taking dry *Spirulina platensis* powder, adding it to PBS buffer or water at approximately pH 7.0, and extracting it at low temperature for 12-36 hours, repeating the swelling process 1-3 times; the low temperature is below 25°C. The freeze-thaw method involves taking dry *Spirulina platensis* powder, adding it to PBS buffer or water at approximately pH 7.0, freezing it for 6-12 hours, repeating the freeze-thaw process 1-3 times. The high-speed homogenization method involves taking dry *Spirulina platensis* powder, adding it to PBS buffer or water at approximately pH 7.0, with the buffer added at a ratio of 10-30 times, extracting at a temperature of 15-60°C, and homogenizing and extracting in 1-3 batches for a total of 30-60 minutes.

[0043] In some embodiments, the centrifugation separation of the extract in the preparation method of Spirulina platensis phycocyanin includes centrifuging the extract after extraction with a centrifuge at a speed >4500 rpm; collecting the supernatant; repeating the extraction of the residue 1-3 times under the same conditions; and combining the supernatants.

[0044] In some embodiments, the purification method for preparing Spirulina platensis phycocyanin includes purifying the supernatant using activated carbon adsorption or salting-out purification. In some specific embodiments, the activated carbon adsorption purification method involves adding activated carbon to the supernatant for 5-30 minutes. The salting-out purification method involves adding salt to the supernatant to 10-40% saturation, allowing it to stand at 2-5°C for salting-out, and centrifuging to collect the precipitate; then adding salt to the supernatant again to 30-70% saturation; allowing it to stand at 2-5°C overnight for salting-out, and centrifuging to collect the precipitate; combining the two precipitates, reconstituted with PBS buffer, and dialysis for desalting.

[0045] In some embodiments, the salt includes ammonium sulfate, sodium sulfate, sodium chloride, ammonium chloride, etc.

[0046] In some embodiments, the preparation method of Spirulina platensis phycocyanin includes the following steps:

[0047] (1) Extraction: Take the dry powder of Spirulina platensis, add PBS buffer or water at about pH 7.0, and extract by swelling at low temperature for 12-36 hours, with 1-3 swelling times;

[0048] (2) Centrifugal separation: After swelling, the extract is centrifuged at a speed of >4500 rpm; the supernatant is collected, and the residue is extracted 1-3 times under the same conditions, and the supernatants are combined.

[0049] (3) Activated carbon adsorption purification: Add activated carbon to the supernatant, and the adsorption time is 5-30 minutes;

[0050] (4) Centrifugal filtration: After adsorption is complete, centrifuge or filter to remove activated carbon and collect the filtrate;

[0051] (5) Drying: The filtrate is freeze-dried under vacuum to obtain phycocyanin.

[0052] In some embodiments, the preparation method of Spirulina platensis phycocyanin includes the following steps:

[0053] (1) Extraction: Take the dry powder of Spirulina platensis, add it to PBS buffer or water at a pH of about 7.0, freeze for 6-12 hours, and freeze and thaw 1-3 times;

[0054] (2) Centrifugal separation: After swelling, the extract is centrifuged at a speed of >4500 rpm; the supernatant is collected, and the residue is extracted 1-3 times under the same conditions, and the supernatants are combined.

[0055] (3) Activated carbon adsorption purification: Add activated carbon to the supernatant, and the adsorption time is 5-30 minutes;

[0056] (4) Centrifugal filtration: After adsorption is complete, centrifuge or filter to remove activated carbon and collect the filtrate;

[0057] (5) Drying: The filtrate is freeze-dried under vacuum to obtain phycocyanin.

[0058] In some embodiments, the preparation method of Spirulina platensis phycocyanin includes the following steps:

[0059] (1) Extraction: High-speed homogenization method is used. Take Spirulina platensis dry powder, add PBS buffer or water with a pH of about 7.0, the amount of buffer added is 10-30 times, the extraction temperature is 15-60℃, and the extraction is carried out in 1-3 times for a total of 30-60 minutes.

[0060] (2) Centrifugation: After homogenization, the extract is centrifuged at a speed of >4500 rpm; the supernatant is collected, and the residue is extracted 1-3 times under the same conditions, and the supernatants are combined.

[0061] (3) Stepwise salting out: Add salt to the supernatant to 10-40% saturation, let it stand at 2-5℃ for salting out, and collect the precipitate by centrifugation; then add salt to the supernatant to 30-70% saturation; let it stand at 2-5℃ overnight for salting out, and collect the precipitate by centrifugation;

[0062] (4) Dialysis desalting: Combine the two precipitates, reconstitute with PBS buffer, and dialyze to desalt;

[0063] (5) Drying: After dialysis, the solution is freeze-dried under vacuum to obtain phycocyanin.

[0064] In some embodiments, the Haematococcus pluvialis astaxanthin microcapsules are obtained by the following method: Haematococcus pluvialis is subjected to cell wall disruption treatment, then mixed with a wall material and emulsified, and finally made into powdered microcapsules by spray drying or other methods.

[0065] In some embodiments, the Haematococcus pluvialis astaxanthin microcapsules are obtained by the following preparation method:

[0066] (1) Haematococcus pluvialis cell wall breaking: Haematococcus pluvialis powder is homogenized under high pressure, with the pressure controlled at 50-80 MPa, or enzymatic hydrolysis is used to break the cell wall, and cellulase and protease are used in combination for 2-4 hours; after breaking the cell wall of Haematococcus pluvialis, Haematococcus pluvialis slurry is obtained.

[0067] (2) Preparation of wall material solution: Mix the wall material with distilled water and stir to dissolve at 50-70°C to prepare the wall material solution;

[0068] (3) Emulsification: Mix the Haematococcus pluvialis slurry after cell wall disruption with the wall material solution, add emulsifier, and homogenize at 15-25 MPa and 35-45℃ for 5-15 minutes to obtain emulsion;

[0069] (4) Spray drying: The emulsion is spray dried;

[0070] (5) Collect the dried powder to obtain Haematococcus pluvialis astaxanthin microcapsules.

[0071] In some embodiments, the cellulase in the method for preparing Haematococcus pluvialis astaxanthin microcapsules accounts for 0.1-0.3% of the mass of Haematococcus pluvialis powder, and the protease accounts for 0.15-0.35% of the mass of Haematococcus pluvialis powder.

[0072] In some embodiments, the wall material in the method for preparing Haematococcus pluvialis astaxanthin microcapsules is selected from at least one of sucrose, maltodextrin, gum arabic, sodium octenyl succinate starch (SSOS), gelatin, and modified starch.

[0073] In some embodiments, the wall material is a ternary compound of sodium octenyl succinate starch (SSOS), maltodextrin, and sucrose (preferably in a mass ratio of 1-3:1:1). This application utilizes the excellent emulsifying properties of SSOS, the film-forming properties of maltodextrin, and the filling and protective effects of sucrose to synergistically form a microcapsule system with fine particle size and the highest centrifugal stability; the microcapsule particle size is ≤200 nm.

[0074] In some embodiments, the solids content in the wall material solution is 20% to 30%.

[0075] In some embodiments, the mass ratio of Haematococcus pluvialis slurry to wall material solution in the method for preparing Haematococcus pluvialis astaxanthin microcapsules is 1:(2-6).

[0076] In some embodiments, the emulsifier used in the preparation method of Haematococcus pluvialis astaxanthin microcapsules is selected from at least one of Tween 80, sucrose ester, and Span. Preferably, the emulsifier is a mixture of Tween 80, sucrose ester, and Span in a weight ratio of 3:3:1.

[0077] In some embodiments, the amount of emulsifier added in the method for preparing Haematococcus pluvialis astaxanthin microcapsules is in a weight ratio of 1:(1-2) to the wall material solution.

[0078] In some embodiments, the emulsion in the preparation method of Haematococcus pluvialis astaxanthin microcapsules is spray-dried, with the inlet temperature set to 100-190°C, the outlet temperature set to 50-90°C, and the feed flow rate set to 50-600 mL / min; preferably, the inlet temperature is 160-190°C and the outlet temperature is 80-90°C.

[0079] In some embodiments, the emulsion in the preparation method of Haematococcus pluvialis astaxanthin microcapsules is spray-dried using a foam spray drying method, in which nitrogen gas is mixed into the emulsion to control the foam density at 0.3–0.5 g / cm³. 3 The inlet temperature of the spray dryer is 100-120℃, and the outlet temperature is 45-55℃.

[0080] In some embodiments, the Haematococcus pluvialis astaxanthin microcapsules have a particle size D50 ≤ 2 μm, and for example, the particle size is 2 μm, 1.9 μm, 1.8 μm, 1.7 μm, 1.6 μm, 1.5 μm, 1.4 μm, 1.3 μm, 1.2 μm, 1.1 μm, 1.0 μm, 0.9 μm, 0.8 μm, 0.7 μm, 0.6 μm, 0.5 μm, 0.4 μm, 0.3 μm, 0.2 μm or 0.1 μm.

[0081] In some embodiments, the encapsulation rate of the Haematococcus pluvialis astaxanthin microcapsules is ≥90%. Examples include 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%.

[0082] In some embodiments, the mass ratio of the compound microalgae active ingredient, taurine, and vitamin C in the composition is (2.5-22):(0.5-5):(0.2-2).

[0083] In some embodiments, the mass ratio of the composite microalgae active ingredient, taurine, and vitamin C in the composition is (3-15):(1.5-4.5):(0.5-1).

[0084] In some embodiments, the mass ratio of the composite microalgae active ingredient, taurine, and vitamin C in the composition is (3-9):(2-4):1.

[0085] In some embodiments, the mass ratio of the composite microalgae active ingredient, taurine, and vitamin C in the composition is 3:2:1, 3:3:1, 3:4:1, 4:2:1, 4:3:1, 4:4:1, 5:2:1, 5:3:1, 5:4:1, 6:2:1, 6:3:1, 6:4:1, 7:2:1, 7:3:1, 7:4:1, 8:2:1, 8:3:1, 8:4:1, 9:2:1, 9:3:1, or 9:4:1.

[0086] The second aspect of this application claims protection for a solution comprising the composition described in the first aspect.

[0087] In some embodiments, the solution includes, but is not limited to, beverages.

[0088] In some embodiments, the mass percentage of the composite microalgae active ingredient in the solution is 0.25-2.2%.

[0089] In some embodiments, the active ingredient of the composite microalgae accounts for 0.2-1.0% of the solution's mass percentage. Examples include 0.2%, 0.3%, 0.4%, 0.5%, 0.6%, 0.7%, 0.8%, 0.9%, or 1.0%.

[0090] In some embodiments, the solution also includes one or more of sweeteners, acidulants, stabilizers, color-protecting stabilizers, and food flavorings.

[0091] In some embodiments, the sweetener comprises 2-7% of the solution by mass. For example, it can be 2%, 3%, 4%, 5%, 6%, or 7%.

[0092] In some embodiments, the sweetener is selected from xylitol, lactitol, sucrose, sucralose, glucose, etc.

[0093] One or more of the following: fructose, erythritol, maltitol, steviol glycosides, mogrosides, and neotame.

[0094] In some embodiments, the sweetener is selected from a combination of erythritol, steviol glycosides and mogrosides to form a compound sweetener with a mass ratio of (60-90):(1-2):1. This combination can achieve zero sugar and low calories, with a full taste and no bitter aftertaste.

[0095] In some embodiments, the mass ratio of the combination of erythritol, steviol glycosides, and mogrosides can be (78-90):(1-2):1. Examples include 78:(1-2):1, 79:(1-2):1, 80:(1-2):1, 81:(1-2):1, 82:(1-2):1, 83:(1-2):1, 84:(1-2):1, 85:(1-2):1, 86:(1-2):1, 87:(1-2):1, 88:(1-2):1, 89:(1-2):1, or 90:(1-2):1.

[0096] In some embodiments, the acidulant accounts for 0.12-0.6% of the solution's mass. Examples include 0.12%, 0.15%, 0.18%, 0.23%, 0.26%, 0.29%, 0.32%, 0.35%, 0.38%, 0.42%, 0.45%, 0.48%, 0.51%, 0.54%, 0.57%, or 0.60%.

[0097] In some embodiments, the acidulant is selected from one or more of citric acid, L-malic acid, phosphoric acid, lactic acid, acetic acid, sodium citrate, sodium dihydrogen phosphate, and sodium hydrogen phosphate.

[0098] In some embodiments, the acidulant is selected from a combination of citric acid, L-malic acid and sodium citrate to form a pH buffer system of compound acidulant with a mass ratio of (2-3):1:(0.5-1). The pH of the combination is stable at 3.6-4.0, taking into account both flavor and phycocyanin stability.

[0099] In some embodiments, the acidulant is selected from citric acid, L-malic acid and sodium citrate in a mass ratio of 2:1:0.5, 2:1:0.6, 2:1:0.7, 2:1:0.8, 2:1:0.9 or 2:1:1.0.

[0100] In some embodiments, the stabilizer accounts for 0.06-0.35% of the solution mass, preferably 0.15-0.25%. Examples include 0.15%, 0.16%, 0.18%, 0.20%, 0.22%, 0.24%, and 0.25%.

[0101] In some embodiments, the stabilizer is selected from one or more of pectin, gellan gum, xanthan gum, sodium carboxymethyl cellulose, carrageenan, and agar.

[0102] In some embodiments, the stabilizer is selected from a combination of sodium carboxymethyl cellulose and xanthan gum to form a compound stabilizer with a mass ratio of (1~2):1, forming a three-dimensional weak gel network to maintain the uniform suspension of microcapsule particles.

[0103] In some embodiments, the color-protecting stabilizer accounts for 0.03-0.18% of the solution by mass. Examples include 0.04%, 0.05%, 0.06%, 0.07%, 0.08%, 0.09%, 0.10%, 0.11%, 0.12%, 0.13%, 0.14%, 0.15%, 0.16%, 0.17%, or 0.18%.

[0104] In some embodiments, the color-protecting stabilizer is selected from one or more of sodium L-ascorbate, sodium phytate, sodium citrate, disodium EDTA, and sodium D-isoascorbate.

[0105] In some embodiments, the color-protecting stabilizer is a combination of sodium L-ascorbate and sodium phytate, forming a compound color-protecting stabilizer. The mass ratio of sodium L-ascorbate to sodium phytate is (3-5):(1-3). Examples include 3:1, 3:2, 3:3, 4:1, 4:2, 4:3, 5:1, 5:2, or 5:3.

[0106] In some embodiments, the amount of the edible flavoring used is 0.05-0.25% of the mass percentage of the solution. Preferably, it is 0.10-0.20%.

[0107] In some embodiments, a beverage comprises the following components in the following weight percentages: 0.25-2.2% complex microalgae active ingredients, 0.05-0.5% taurine, 0.02-0.2% vitamin C, 2-7% sweetener, 0.12-0.6% acidulant, 0.06-0.35% stabilizer, 0.03-0.18% color-protecting stabilizer, 0.05-0.25% flavoring, with the balance being purified water.

[0108] In some embodiments, a beverage comprises the following components in the following weight percentages: Chlorella vulgaris CGF extract 0.1-1%, Spirulina platensis phycocyanin 0.1-0.7%, Haematococcus pluvialis astaxanthin microcapsules 0.05-0.5%, taurine 0.05-0.5%, vitamin C 0.02-0.2%, sweetener 2-7%, acidulant 0.12-0.6%, stabilizer 0.06-0.35%, color stabilizer 0.03-0.18%, flavoring 0.05-0.25%, and the balance being purified water.

[0109] In some embodiments, a beverage comprises the following components in the following weight percentages: Chlorella vulgaris CGF extract 0.4-0.8%, Spirulina platensis phycocyanin 0.3-0.6%, Haematococcus pluvialis astaxanthin microcapsules 0.2-0.4%, taurine 0.2-0.5%, vitamin C 0.05-0.15%, sweetener 5-7%, acidulant 0.3-0.6%, stabilizer 0.15-0.3%, color stabilizer 0.07-0.15%, edible flavoring 0.12-0.15%, and the balance being purified water.

[0110] A third aspect of this application provides a method for preparing a solution, comprising the following steps:

[0111] (1) Preparation of compound microalgae active ingredients: The extract of Chlorella pulvinata CGF and phycocyanin of Spirulina platensis were added to a portion of room temperature pure water and stirred until completely dissolved. The mixture was filtered through a microporous membrane to remove impurities and obtain water-soluble microalgae active liquid. Astaxanthin microcapsules of Haematococcus pluvialis were added to water and stirred at low speed to disperse evenly to obtain astaxanthin microcapsule suspension.

[0112] (2) Preparation of colloidal solution: The stabilizer and sweetener are pre-dry mixed evenly, added to water, stirred at high speed until completely swollen and dissolved, cooled to obtain colloidal solution;

[0113] (3) Nutritional preparation: Add acidulant, color stabilizer and taurine to colloidal solution in sequence and stir until completely dissolved; then slowly add water-soluble microalgae active liquid and astaxanthin microcapsule suspension, stir evenly and then add vitamin C and edible flavoring, add purified water to make up the volume to obtain the liquid.

[0114] (4) Homogenization and degassing: The liquid feed is homogenized and degassed under high pressure;

[0115] (5) Instant sterilization and filling.

[0116] In some embodiments, the water temperature is 35~40°C when the Haematococcus pluvialis astaxanthin microcapsules described in step (1) are added.

[0117] In some implementations, the water temperature in step (2) is 60~65°C.

[0118] In some embodiments, the homogenization temperature in step (4) is 40~45℃ and the pressure is 20~30MPa.

[0119] In some embodiments, the pore size of the microporous filter membrane in step (1) is 0.22-0.45 μm.

[0120] In some implementations, the instantaneous sterilization conditions in step (5) are a temperature of 135~138℃ and a time of 3~5s.

[0121] In some embodiments, the vitamin C in step (3) is added at a low temperature before homogenization; the low temperature is no higher than 25°C.

[0122] In some embodiments, the preparation method is carried out under light-protected conditions to protect the activity of phycocyanin and astaxanthin.

[0123] In some embodiments, a method for preparing a solution includes the following steps:

[0124] (1) Preparation of microalgae active phase: The CGF extract of Chlorella pulvinata and phycocyanin of Spirulina platensis were added to a portion of room temperature pure water and stirred until completely dissolved. The mixture was filtered through a 0.22 μm microporous membrane to remove impurities and obtain a water-soluble microalgae active liquid. Astaxanthin microcapsules of Haematococcus pluvialis were added to warm water at 35~40℃ and stirred at low speed to disperse evenly to obtain an astaxanthin microcapsule suspension.

[0125] (2) Preparation of colloidal solution: The composite stabilizer and composite sweetener are pre-dry mixed evenly, added to pure water at 60-65℃, stirred at high speed until completely swollen and dissolved, and cooled to below 40℃ to obtain colloidal solution;

[0126] (3) Nutritional preparation: Add acidulant, color stabilizer and taurine to colloidal solution in sequence and stir until completely dissolved; then slowly add water-soluble microalgae active liquid and astaxanthin microcapsule suspension, stir evenly and then add vitamin C and edible flavoring, add purified water to make up the volume to obtain the liquid.

[0127] (4) Homogenization and degassing: The liquid is heated to 40~45℃ and homogenized twice under high pressure of 20~30MPa. Then it is degassed for 5~10min under vacuum of -0.07~-0.08MPa to remove dissolved oxygen and reduce oxidative degradation.

[0128] (5) Instant sterilization and filling: Ultra-high temperature instant sterilization is adopted. The sterilization conditions are 135~138℃ for 3~5s. After sterilization, it is quickly cooled to below 25℃. Filling and sealing are carried out in an aseptic environment. After passing the light protection inspection, the finished product is obtained.

[0129] The fourth aspect of this application provides the use of the composition described in the first aspect or the solution described in the second aspect in the preparation of antioxidant and anti-fatigue products.

[0130] In some implementations, the product includes, but is not limited to, food, health products, or pharmaceuticals.

[0131] This application has the following beneficial effects:

[0132] 1. The formula in this application constructs a four-dimensional anti-fatigue system of "energy supply - metabolic acceleration - oxidative protection - tissue repair". Taurine replenishes energy and maintains myocardial function; Vitamin C promotes collagen synthesis and accelerates the removal of metabolic waste; phycocyanin removes lactic acid and reduces inflammatory response; astaxanthin protects mitochondrial membranes and reduces oxidative damage; Chlorella CGF promotes cell proliferation and repair and enhances the body's endurance. The five components work synergistically and complement each other. In a mouse weight-bearing swimming test, the time to exhaustion was increased by more than 50% compared to traditional taurine and Vitamin C drinks.

[0133] 2. Water-oil dual-phase antioxidant protection for more thorough free radical scavenging: Vitamin C, phycocyanin, and CGF form a water-soluble antioxidant defense line, acting on the cytoplasm and extracellular fluid; astaxanthin microcapsules form a lipid-soluble antioxidant defense line, penetrating the cell membrane to act on the lipid bilayer and mitochondria. Together, they construct a whole-cell protection system, increasing DPPH free radical scavenging rate by more than 45% compared to single antioxidant systems.

[0134] 3. Multiple steady-state technologies ensure long shelf-life activity: Microencapsulation technology isolates astaxanthin from the aqueous oxidative environment; a compound color-protecting stabilizer protects phycocyanin and vitamin C through a triple mechanism of chelating metal ions, removing free oxygen, and buffering pH; combined with instant sterilization and vacuum degassing processes, the product can be stored at room temperature away from light for 6 months with a core active ingredient retention rate of ≥82% and no significant color change, solving the industry pain point of short shelf life of microalgae active ingredients.

[0135] 4. Only astaxanthin microcapsules encapsulated in the above-mentioned wall material system can be stably dispersed and effectively released in the aqueous phase of beverages, forming a complete water-oil dual-phase protection system together with CGF and phycocyanin (aqueous phase antioxidant). Unencapsulated astaxanthin oil is basically degraded in beverages within 3 months (as confirmed by Experimental Example 2 and Comparative Example 2).

[0136] 5. Fresh and palatable flavor, zero sugar and healthier: Through three methods—optimized sweet and sour buffer system, microencapsulation to mask fishy smell, and blending of natural sweeteners to enhance flavor—the product effectively masks the characteristic fishy smell of microalgae, resulting in a fresh and natural flavor. It uses a zero-sugar formula, is low in calories, and conforms to the current trend of healthy consumption.

[0137] 6. Strong process controllability and easy industrial production: The preparation process complies with food safety production standards, is fully compatible with existing beverage production lines, and requires no additional special equipment; the process parameters are precise and controllable, the activity retention rate is high, and it is suitable for large-scale mass production. Attached Figure Description

[0138] Figure 1 This is a flowchart illustrating the preparation process of the functional beverage described in this application. Detailed Implementation

[0139] To enable those skilled in the art to better understand the technical solutions of this application, the application will be further described in detail below with reference to the accompanying drawings and specific embodiments. It should be understood that the specific embodiments described herein are only used to explain this application and represent a part of the embodiments of this application, not all of them. All other embodiments obtained by those skilled in the art based on the embodiments of this application without creative effort are within the protection scope of this application.

[0140] I. Preparation Examples

[0141] Example 1: Preparation method of Chlorella vulgaris CGF extract

[0142] (1) Low temperature soaking: Mix Chlorella pyrenoidosa powder and water at a weight ratio of 1:20 and stir evenly. Soak at a low temperature of 4-10℃ for 2 hours.

[0143] (2) Steam thermal swelling and cell wall breaking treatment: The soaked algal solution was subjected to cell wall breaking treatment by using multiple streams of steam at different pressures and temperatures to simultaneously perform thermal swelling and cell wall breaking treatment on Chlorella. Specifically, one stream was at a pressure of 150 Pa and a temperature of 100 ℃, another stream was at a pressure of 180 Pa and a temperature of 110 ℃, and yet another stream was at a pressure of 200 Pa and a temperature of 120 ℃. This process was repeated three times, with a total treatment time of 100 minutes, to obtain cell wall broken cytoplasm.

[0144] (3) Hot water extraction: After the cell wall is broken, a 1000L reactor is used as the extraction container. 350L of cell wall broken cytoplasm is added to the reactor, and 350L of water is added. The reaction temperature is 45±2℃, the stirring speed is 150rpm, and the extraction time is 120 minutes. During the extraction reaction, deoxyribonuclease is added at a rate of 7 million units / L of reaction solution.

[0145] (4) Centrifugal separation: After extraction, centrifuge the sample using a disc centrifuge at a speed of >4500 rpm;

[0146] (5) Ultrafiltration treatment: The supernatant obtained by centrifugation is subjected to ultrafiltration treatment. The molecular weight cutoff of the ultrafiltration membrane is 3kDa, and the working conditions are 25-45℃. The ultrafiltration adopts a hollow fiber ultrafiltration system with a membrane pore size of 0.1μm.

[0147] (6) Concentration and drying: Collect the ultrafiltration concentrate and dry it using a vacuum drying tower with a vacuum degree of 0.01 MPa and a temperature of 50 ± 2℃; after drying, Chlorella proteinensis CGF dry powder is obtained.

[0148] The ultrafiltration concentrate described in step (6) has a solids content ≤3% and an OD content ≤3%. 260 nm≥100.

[0149] The total mass content of nucleotides and amino acids in the Chlorella CGF dry powder was found to be 49.39%.

[0150] Example 2: Preparation method of astaxanthin microcapsules from Haematococcus pluvialis (1) Haematococcus pluvialis cell wall breaking: Haematococcus pluvialis powder is homogenized under high pressure and the pressure is controlled at 50-80 MPa to obtain Haematococcus pluvialis slurry; (2) Preparation of wall material solution: The wall material is mixed with distilled water and stirred at 50-70°C to dissolve it, thus preparing a wall material solution; the wall material is a mixture of sodium octenyl succinate starch (SSOS), maltodextrin and sucrose (mass ratio 3:1:1); the solid content of the wall material solution is 20%-30%; (3) Emulsification: The Haematococcus pluvialis slurry after cell wall disruption is mixed with the wall material solution, and the weight ratio of the wall material solution to the Haematococcus pluvialis slurry is 4:1; the emulsifier is added at a weight ratio of 1:1 with the wall material solution, and the mixture is homogenized for 10 minutes at 15-25 MPa and 35-45℃ to obtain an emulsion; the emulsifier is Tween 80, sucrose ester and Span mixed at a weight ratio of 3:3:1. (4) Spray drying: The emulsion is spray dried with the inlet temperature set to 160-190℃ and the outlet temperature set to 80-90℃; the feed flow rate is 300-400mL / min; spray drying is carried out until the wall material condenses to obtain Haematococcus pluvialis astaxanthin microcapsule powder; the particle size of the Haematococcus pluvialis astaxanthin microcapsule is 188.10 nm.

[0151] Example 3: Preparation method of astaxanthin microcapsules from Haematococcus pluvialis

[0152] (1) Cell wall disruption of Haematococcus pluvialis: Haematococcus pluvialis powder was subjected to enzymatic hydrolysis to disrupt the cell wall. The powder was treated with a combination of cellulase and protease for 2-4 hours to obtain Haematococcus pluvialis slurry. The cellulase content was 0.15% of the mass of Haematococcus pluvialis powder and the protease content was 0.18% of the mass of Haematococcus pluvialis powder. The protease activity was 30 U / mg.

[0153] (2) Preparation of wall material solution: The wall material is mixed with distilled water and stirred and dissolved at 50-70°C to prepare a wall material solution; the wall material is a mixture of sodium octenyl succinate starch (SSOS), maltodextrin and sucrose (mass ratio 3:1:1); the solid content of the wall material solution is 20%-30%.

[0154] (3) Emulsification: The Haematococcus pluvialis slurry after cell wall disruption is mixed with the wall material solution, and the weight ratio of the wall material to the Haematococcus pluvialis slurry is 4:1; the emulsifier is added to the wall material solution at a weight ratio of 1:1, and the mixture is homogenized for 10 minutes at 15-25 MPa and 35-45℃ to obtain an emulsion; the emulsifier is Tween 80, sucrose ester and Span mixed at a weight ratio of 3:3:1.

[0155] (4) Spray drying: The emulsion is spray dried with the inlet temperature set to 160-190℃ and the outlet temperature set to 80-90℃; the feed flow rate is 300-400mL / min; spray drying is carried out until the wall material condenses to obtain Haematococcus pluvialis astaxanthin microcapsule powder; the particle size of the Haematococcus pluvialis astaxanthin microcapsule is 200nm.

[0156] Example 4: Preparation method of astaxanthin microcapsules from Haematococcus pluvialis

[0157] (1) Haematococcus pluvialis cell wall breaking: Haematococcus pluvialis powder is homogenized under high pressure and the pressure is controlled at 50-80 MPa to obtain Haematococcus pluvialis slurry;

[0158] (2) Preparation of wall material solution: The wall material is mixed with distilled water and stirred at 50-70°C to dissolve it, thus preparing a wall material solution; the wall material is a mixture of sodium octenyl succinate starch (SSOS), maltodextrin and sucrose (weight ratio 3:1:1); the solid content of the wall material solution is 20%-30%;

[0159] (3) Emulsification: The Haematococcus pluvialis slurry after cell wall disruption is mixed with the wall material solution, and the weight ratio of the wall material to the Haematococcus pluvialis slurry is 4:1; the emulsifier is added to the wall material solution at a weight ratio of 1:1, and the mixture is homogenized for 10 minutes at 15-25 MPa and 35-45℃ to obtain an emulsion; the emulsifier is Tween 80, sucrose ester and Span mixed at a weight ratio of 3:3:1.

[0160] (4) Spray drying: Foam spray drying method is adopted, that is, nitrogen gas is mixed into the emulsion to control the foam density at 0.3-0.5 g / cm³. 3 The inlet temperature of the spray dryer is 100-120℃, and the outlet temperature is 45-55℃; the particle size of the Haematococcus pluvialis astaxanthin microcapsules is 195nm.

[0161] Example 5: Preparation method of phycocyanin from Spirulina platensis

[0162] (1) Extraction: Take the dry powder of Spirulina platensis, add PBS buffer at pH 7.0, the material-to-liquid ratio is 1:50 (g / mL), and extract by swelling at 15℃ for 24 hours, and repeat the swelling twice;

[0163] (2) Centrifugation: After swelling is complete, the extract is centrifuged at 5000 rpm for 20 minutes and the supernatant is collected; the residue is extracted once under the same conditions.

[0164] (3) Activated carbon adsorption purification: Add 300 mesh activated carbon to the supernatant, the amount of activated carbon added is 0.7g / 20mL of extract, and the adsorption time is 10 minutes;

[0165] (4) Centrifugal filtration: After adsorption is complete, centrifuge or filter to remove activated carbon and collect the filtrate;

[0166] (5) Drying: The filtrate is freeze-dried under vacuum to obtain phycocyanin.

[0167] Tests showed that the purity of phycocyanin can reach 80%.

[0168] Example 6: Preparation method of phycocyanin from Spirulina platensis

[0169] (1) Extraction: Take the dry powder of Spirulina platensis, add PBS buffer at pH 7.0, the material-to-liquid ratio is 1:50 (g / mL), freeze for 8 hours, and freeze and thaw twice;

[0170] (2) Centrifugation: After freeze-thaw, centrifuge at 5000 rpm for 20 minutes and collect the supernatant; the residue is extracted once under the same conditions.

[0171] (3) Activated carbon adsorption purification: Add 300 mesh activated carbon to the supernatant, the amount of activated carbon added is 0.7g / 20mL of extract, and the adsorption time is 10 minutes;

[0172] (4) Centrifugal filtration: After adsorption is complete, centrifuge or filter to remove activated carbon and collect the filtrate;

[0173] (5) Drying: The filtrate is freeze-dried under vacuum to obtain phycocyanin.

[0174] Example 7: Preparation method of phycocyanin from Spirulina platensis

[0175] (1) Extraction: The high-speed homogenization method was used. Spirulina platensis powder was added to PBS buffer or water at a pH of about 7.0. The amount of buffer added was 20 times. The extraction temperature was 30℃. The extraction was carried out in 3 homogenizations for a total of 40 minutes.

[0176] (2) Centrifugation: After homogenization, centrifuge at 5000 rpm for 20 minutes and collect the supernatant; the residue is extracted once under the same conditions.

[0177] (3) Stepwise salting out: Add ammonium sulfate to the supernatant to 30% saturation, let it stand at 4°C for salting out, and collect the precipitate by centrifugation; then add ammonium sulfate to the supernatant to 50% saturation; let it stand at 4°C for salting out overnight, and collect the precipitate by centrifugation;

[0178] (4) Dialysis desalting: Combine the two precipitates, reconstitute them with PBS buffer, and dialyze to desalt;

[0179] (5) Drying: The dialysis solution is freeze-dried under vacuum to obtain phycocyanin.

[0180] The purity of phycocyanin was found to be 85.6%.

[0181] Example 8: Functional Beverage Preparation Method

[0182] (1) Preparation of microalgae active phase: The extract of Chlorella pulvinata CGF and phycocyanin of Spirulina platensis were added to 10% of the formula amount of room temperature pure water and stirred until completely dissolved. The solution was filtered through a 0.22μm microporous membrane to remove impurities and obtain water-soluble microalgae active liquid. Astaxanthin microcapsules of Haematococcus pluvialis were added to 5% of the formula amount of 38℃ warm water and stirred at low speed to disperse evenly to obtain astaxanthin microcapsule suspension.

[0183] (2) Preparation of colloidal solution: Sodium carboxymethyl cellulose, xanthan gum, erythritol, steviol glycosides and mogrosides are pre-dry mixed evenly, added to 60℃ pure water, stirred at high speed for 15 min until completely swollen and dissolved, and cooled to 38℃ to obtain colloidal solution;

[0184] (3) Nutritional preparation: Add citric acid, L-malic acid, sodium citrate, sodium L-ascorbate, sodium phytate and taurine to the colloidal solution in sequence and stir until completely dissolved; then slowly add water-soluble microalgae active liquid and astaxanthin microcapsule suspension, stir evenly and then add vitamin C and grapefruit flavoring, add purified water to make up the volume to obtain the solution;

[0185] (4) Homogenization and degassing: The liquid material is heated to 42℃ and homogenized twice under high pressure of 25MPa, and then degassed for 8 minutes under vacuum of -0.075MPa.

[0186] (5) Instant sterilization and filling: Ultra-high temperature instant sterilization is adopted. The sterilization conditions are 137℃ and 4s. After sterilization, it is rapidly cooled to 22℃, filled and sealed in an aseptic environment, and the finished product is obtained after passing the light-proof inspection.

[0187] II. Functional Beverage Formulation Examples

[0188] Examples A1-A7: The formulas for functional beverages are shown in Table 1.

[0189] Preparation method:

[0190] (1) Take the protein-nucleated Chlorella CGF extract prepared in Example 1, the Spirulina platensis phycocyanin prepared in Example 7, and the Haematococcus pluvialis astaxanthin microcapsules prepared in Example 2 for later use;

[0191] (2) The functional beverage was prepared according to the preparation method in Example 8.

[0192] Table 1. Functional Beverage Formulas and Properties

[0193] III. Experimental Examples Experimental Example 1: In vitro antioxidant activity assay Experimental group: Formulation of Example A2 Comparative Example 1: Formula without complex microalgae active ingredients Preparation: Without the complex microalgae active ingredients, water was added to bring the total to 100%. The content and preparation method of the remaining taurine, vitamin C and matrix components were the same as in Example A2.

[0194] Comparative Example 2: Formula containing Spirulina platensis + Taurine + Vitamin C Preparation: Only 0.9% Spirulina platensis powder was added to replace the active ingredients of the compound microalgae. The remaining components, contents, and preparation methods were the same as in Example A2.

[0195] Comparative Example 3: Formula containing Chlorella vulgaris CGF extract + taurine + vitamin C Preparation: Only 0.9% of Chlorella vulgaris CGF extract was added to replace the active ingredients of the compound microalgae. The remaining components, contents, and preparation methods were the same as in Example A2.

[0196] Comparative Example 4: Formula containing Haematococcus pluvialis astaxanthin microcapsules + taurine + vitamin C Preparation: Only 0.9% Haematococcus pluvialis astaxanthin microcapsules were added to replace the active ingredients of the complex microalgae. The remaining components, contents, and preparation methods were the same as in Example A2.

[0197] Comparative Example 5: Formula containing Spirulina platensis powder + Chlorella vulgaris powder + Haematococcus pluvialis powder Preparation: Spirulina platensis raw powder 0.3%, Chlorella proteoglycans raw powder 0.2%, Haematococcus pluvialis raw powder 0.2%, and the remaining components and contents are the same as in Example A2.

[0198] Functional beverage preparation method: (1) Preparation of active phase of microalgae: Add Chlorella pyrenoidosa powder and Spirulina platensis powder to 10% of the formula amount of room temperature pure water to obtain active ingredient 1; add Haematococcus pluvialis powder to 5% of the formula amount of 38℃ warm water and stir at low speed to disperse evenly to obtain active ingredient II; (2) Preparation of colloidal solution: Xanthan gum, sodium carboxymethyl cellulose, erythritol, steviol glycosides and mogrosides are pre-dry mixed evenly, added to 60℃ pure water, stirred at high speed for 15 min until completely swollen and dissolved, and cooled to 38℃ to obtain colloidal solution; (3) Nutritional preparation: Add citric acid, L-malic acid, sodium citrate, sodium L-ascorbate, sodium phytate and taurine to the colloidal solution in sequence and stir until completely dissolved; then slowly add active ingredient I and active ingredient II, stir evenly, add vitamin C and grapefruit flavoring, add purified water to make up the volume, and obtain the liquid. (4) Homogenization and degassing: The liquid material is heated to 42℃ and homogenized twice under high pressure of 25MPa, and then degassed for 8 minutes under vacuum of -0.075MPa. (5) Instant sterilization and filling: Ultra-high temperature instant sterilization is adopted. The sterilization conditions are 137℃ and 4s. After sterilization, it is rapidly cooled to 22℃, filled and sealed in an aseptic environment, and the finished product is obtained after passing the light-proof inspection.

[0199] Experimental method: In vitro antioxidant activity assay The antioxidant capacity of each sample was determined using the DPPH free radical scavenging method, and the scavenging rate was determined at the same dilution factor.

[0200] The DPPH free radical scavenging method shall be performed in accordance with GB / T 39100-2020.

[0201] Results: See Table 2 Table 2. DPPH free radical scavenging rate of different samples

[0202] The results showed that the antioxidant activity of the compound formulation in this application was significantly better than that of the comparative formulation, verifying that the multi-active components of the compound microalgae after specific extraction have a synergistic effect with taurine and vitamin C in terms of antioxidant properties.

[0203] Experimental Example 2: Anti-fatigue test in mice Groups: The experimental group and the control group are the same as in Experiment 1.

[0204] Experimental methods: Eighty SPF-grade male Kunming mice were randomly divided into eight groups: blank control group, comparative example 1, comparative example 2, comparative example 3, comparative example 4, comparative example 5, low-dose group of Example A2, and high-dose group of Example A2. The corresponding samples were administered by gavage daily for 30 consecutive days. After gavage, a weight-bearing swimming test (5% of body weight) was performed. The time to exhaustion was recorded, and the blood lactate and liver glycogen content after exercise were measured. The results are shown in Table 3.

[0205] Table 3 Results of mouse anti-fatigue test

[0206] The results showed that the beverage of this application could significantly prolong the exhaustive swimming time of mice, reduce blood lactate levels after exercise, and increase liver glycogen reserves. Moreover, the effect was significantly better than that of traditional taurine VC beverages and single microalgae formulations, demonstrating the superiority of multi-component synergistic anti-fatigue effects.

[0207] Test Example 3: Accelerated Stability Test Experimental group: Example A2 Comparative Example 1: Formulation without color-protecting stabilizer Preparation: Without the composite color-protecting stabilizer (L-sodium ascorbate, sodium phytate), water was added to bring the total to 100%. The remaining components, contents, and preparation methods were the same as in Example A2.

[0208] Comparative Example 2: Haematococcus pluvialis oil (unencapsulated in microcapsules) formulation Except for replacing the Haematococcus pluvialis astaxanthin microcapsules with Haematococcus pluvialis oil (unencapsulated) of the same effective content, the other components and preparation methods are completely consistent with Example A2.

[0209] Preparation of Haematococcus pluvialis oil: (1) Haematococcus pluvialis cell wall breaking: Haematococcus pluvialis powder is homogenized under high pressure and the pressure is controlled at 50-80 MPa to obtain Haematococcus pluvialis slurry; (2) Emulsification: Add emulsifier to Haematococcus pluvialis slurry and homogenize for 10 minutes at 15-25 MPa and 35-45℃ to obtain Haematococcus pluvialis oil.

[0210] Comparative Example 3: Conventional Pasteurization Preparation: Except for replacing the sterilization method in Example A2 with pasteurization (65°C, 30 min), the other components, contents, and preparation methods are the same as in Example A2.

[0211] Experimental method: The samples of Example 2 and Comparative Examples 1-3 were stored in the dark at 37°C and 75% relative humidity for 3 months. The retention rates of phycocyanin, astaxanthin and vitamin C were measured periodically, and the appearance changes were observed. The results are shown in Table 4.

[0212] Table 4. Retention rate of active ingredients after 3 months of accelerated testing

[0213] As shown in Table 4, after 3 months of accelerated testing at 37°C, Example A2 showed that the phycocyanin retention rate was 84.6%, the astaxanthin retention rate was 82.1%, and the vitamin C retention rate was 88.3%. The product had a uniform color and no sedimentation or stratification.

[0214] The results demonstrate that the composite color-protecting stabilizer, microencapsulation technology, and instant sterilization of this application can significantly improve the stability of each active ingredient during its shelf life and effectively delay oxidative degradation and fading.

[0215] Experiment Example 4: Sensory Evaluation Experiment Experimental group: Example A2 Comparative Example 2: The same formulation as Comparative Example 1, Haematococcus pluvialis oil (not encapsulated in microcapsules). Twenty professional sensory evaluators were selected to conduct sensory evaluations of Example A2 and Comparative Example 2 on a 10-point scale. The evaluation indicators included flavor, color, taste, and overall acceptability. The average value was taken, and the results are shown in Table 5.

[0216] Table 5 Sensory Evaluation Results

[0217] The results show that this application effectively masks the characteristic fishy smell of microalgae through microencapsulation optimization, and significantly improves the sensory quality of the product.

[0218] Experimental Example 5: Screening Test for Color-Protecting Stabilizers To screen suitable color-protecting and stabilizer combinations for complex microalgae beverage systems, the effects of different color-protecting agents and their compound systems on the color stability of the beverage were investigated. Based on the formulation of Example A2 (without color-protecting and stabilizer), different color-protecting agents were added, and the mixtures were stored in the dark at 37°C and 75% relative humidity for 3 months.

[0219] The total color difference value and browning inhibition rate were used as evaluation indicators, and the results are shown in Table 6.

[0220] Table 6. Effects of different color-protecting agent systems on the color stability of compound microalgae beverages (37℃, 3 months)

[0221] Table 6 shows that the blank control (D1), without color-protecting agent treatment, had a ΔE of 12.8 ± 1.2 after 3 months, indicating severe color deterioration. Among the single color-protecting agents, sodium L-ascorbate and sodium phytate both showed good color-protecting effects, exhibiting a certain dose-dependent effect. When the addition amount increased from 0.02% to 0.05%, ΔE decreased significantly; further increasing to 0.10% resulted in a slower improvement (D3 vs D4, D6 vs D7). Considering both effectiveness and cost, the optimal addition amount of a single color-protecting agent was 0.05%.

[0222] When sodium L-ascorbate and sodium phytate are used in a 1:1 ratio (D8–D10), the color-protecting effect is significantly better than that of a single color-protecting agent. The optimal effect is achieved when the total addition is 0.10% (0.05% each). The color intensity was only 3.5 ± 0.5, with a browning inhibition rate of 72.7% and a sensory color score of 8.5. Studies have shown that the combined use of ascorbic acid and sodium phytate has a significant synergistic antioxidant effect; ascorbic acid can slow down the Maillard reaction and reduce the degradation of carotenoids and phenolic substances; sodium phytate inhibits oxidation reactions by chelating metal ions. The combination of these two substances can synergistically exert color-protecting effects through two different pathways: free radical scavenging and metal ion chelation.

[0223] The effects of combining sodium L-ascorbate with EDTA-2Na (D11) or sodium phytate with EDTA-2Na (D12) are not as good as those of combining sodium L-ascorbate with sodium phytate (D9).

[0224] In summary, the optimal color-protecting stabilizer for compound microalgae beverages is a mixture of sodium L-ascorbate and sodium phytate in a 1:1 weight ratio, with a total addition of 0.08% to 0.12%, preferably 0.10% (0.05% each).

[0225] Experimental Example 6: Stabilizer Screening Test To screen suitable stabilizer combinations for complex microalgae beverage systems, the effects of different stabilizers and their compound systems on the suspension stability of the beverage were investigated. Based on the formulation of Example A2 (without added stabilizers), different stabilizers were added, and the centrifugal sedimentation rate and the stratification after standing at room temperature for 30 days were used as evaluation indicators. The results are shown in Table 7 below.

[0226] Table 7. Effects of stabilizer system on the stability and taste of functional beverages (37℃, 30 days)

[0227] As shown in Table 7, the blank control (E1) without stabilizer treatment had a centrifugation sedimentation rate of 8.6±0.9%, and obvious stratification occurred within 30 days.

[0228] Among single stabilizers, CMC-Na (E2-E4), xanthan gum (E5-E7), and gellan gum (E8-E10) can all reduce centrifugal sedimentation rates to some extent. CMC-Na can prevent stratification at concentrations above 0.20%, but excessively high concentrations (0.30%) result in a thicker, more mushy texture. Xanthan gum can prevent stratification at 0.05% concentration, exhibiting excellent suspension properties. Gellan gum can also prevent stratification at 0.05% concentration, while maintaining a refreshing taste.

[0229] When a compound stabilizer is used, suspension stability is further improved. The optimal effect (E12) is achieved when CMC-Na and xanthan gum are combined in a 1:1 ratio, with a total addition of 0.20% (0.10% each), resulting in a centrifugal sedimentation rate of only 2.1±0.3%, no stratification after 30 days, and a smooth mouthfeel. Research indicates that the combination of CMC-Na and xanthan gum can effectively suspend microcapsules and particles in beverages through a dual mechanism of synergistic thickening and the formation of a three-dimensional network structure.

[0230] The effects of CMC-Na combined with gellan gum (E13) or xanthan gum combined with gellan gum (E14) are slightly inferior to those of CMC-Na combined with xanthan gum. Although the ternary combination of the three stabilizers (E15) has good stability, the taste is slightly thicker and not as refreshing as the binary combination.

[0231] In summary, the optimal stabilizer for compound microalgae beverages is a mixture of sodium carboxymethyl cellulose and xanthan gum in a 1:1 weight ratio, with a total addition of 0.15% to 0.25%, preferably 0.20% (0.10% each).

[0232] Experimental Example 7: Extraction Method of CGF from Chlorella proteoglycans This experiment systematically optimized the extraction process of Chlorella vulgaris CGF from five dimensions: cell wall disruption method, extraction temperature, extraction time, material-liquid ratio, and molecular weight cutoff of ultrafiltration membrane.

[0233] Table 8. Investigation of cell wall disruption methods and extraction temperature and time of Chlorella proteoglycans

[0234] Note: Steam thermal expansion is divided into three steam streams (150Pa / 100℃, 180Pa / 110℃, 200Pa / 120℃); one steam stream thermal expansion is 180Pa / 110℃.

[0235] This experiment, through system optimization, determined the optimal extraction process as follows: steam thermal expansion and cell wall disruption (co-operation of multiple steam streams at different pressures and temperatures) + 45±2℃ hot water extraction + 3kDa ultrafiltration, maximizing the CGF extraction rate (OD). 260 The nanometer count (nm) reached over 110, significantly superior to conventional high-pressure homogenization (OD). 260 nm=82.6) and ultrasonic fragmentation (OD) 260 nm89.4).

[0236] Experiment Example 8: Screening Experiment of Astaxanthin Microcapsule Wall Material from Haematococcus pluvialis This experiment systematically investigated the effects of different wall materials and combinations thereof on the encapsulation efficiency, drug loading, and long-term stability of astaxanthin microcapsules.

[0237] Table 9. Effects of different wall materials and combinations thereof on astaxanthin microcapsule encapsulation efficiency, drug loading, and long-term stability.

[0238] Astaxanthin, being a fat-soluble component, is readily oxidized and degraded in aqueous beverages. This experiment, through wall material screening, determined the optimal wall material system to be a ternary compound of sodium octenyl succinate starch (SSOS), maltodextrin, and sucrose (3:1:1), achieving an encapsulation rate of 92.85% and an astaxanthin retention rate of 84.7% after 6 months.

[0239] Experimental Example 9: Screening of the Dosage of Compound Microalgae Active Ingredients in Beverages To determine the optimal addition amount of the compound microalgae active ingredients in beverages, based on the formulation of Example A2 (without the compound microalgae active ingredients), different proportions of the compound microalgae active ingredients (with Chlorella vulgaris CGF extract: Spirulina platensis phycocyanin: Haematococcus pluvialis astaxanthin microcapsules = 1:1:1) were added. The DPPH free radical scavenging rate, sensory score, and 3-month stability were used as evaluation indicators. The results are shown in Table 10 below.

[0240] Table 10 DPPH radical scavenging rate, sensory score and 3-month stability of different samples

[0241] As shown in Table 10, the DPPH free radical scavenging rate continued to rise with the increase of the amount of compound microalgae active ingredients. When the amount of added ingredients increased from 0 to 0.9%, the scavenging rate increased significantly from 18.3% to 84.5%; when it continued to increase to 1.2%, the increase in scavenging rate slowed down (only increasing to 86.1%).

[0242] However, sensory flavor and color scores decreased with increasing addition amount. When the addition amount exceeded 0.9%, the characteristic flavor of microalgae began to emerge, the color became too dark, and significant sedimentation appeared after 3 months. Studies have shown that higher phycocyanin concentrations result in more stable beverages, but excessively high concentrations can affect taste and color.

[0243] Considering antioxidant activity, sensory quality, and storage stability, the optimal addition amount of the complex microalgae active ingredient (Chlorella vulgaris CGF extract: Spirulina platensis phycocyanin: Haematococcus pluvialis astaxanthin microcapsules = 1:1:1) is 0.7% to 1.0% of the total beverage mass, preferably 0.9%. At this addition amount, the DPPH scavenging rate reaches 84.5%, the sensory quality is good, and only slight precipitation occurs during the 3-month storage period, demonstrating optimal overall performance.

[0244] Preferably, the amount of the compound microalgae active ingredients added to the beverage is 0.7% to 1.0%.

[0245] More preferably, the amount of the compound microalgae active ingredient added is 0.9%.

[0246] It is understood that the above embodiments are merely exemplary implementations used to illustrate the principles of this application, and this application is not limited thereto. For those skilled in the art, various modifications and improvements can be made without departing from the spirit and substance of this application, and these modifications and improvements are also considered to be within the scope of protection of this application.

Claims

1. A composition, characterized in that, The composition comprises a complex microalgae active ingredient, taurine, and vitamin C, wherein the mass ratio of the complex microalgae active ingredient, taurine, and vitamin C is (2.5-22):(0.5-5):(0.2-2); wherein the complex microalgae active ingredient is composed of Chlorella CGF extract, Spirulina phycocyanin, and Haematococcus pluvialis astaxanthin microcapsules.

2. The composition according to claim 1, characterized in that, The composite microalgae active ingredient is composed of 1-8 parts by weight of Chlorella CGF extract, 1-7 parts by weight of Spirulina phycocyanin, and 0.5-5 parts by weight of Haematococcus pluvialis astaxanthin microcapsules; preferably, the composite microalgae active ingredient is composed of 2-6 parts by weight of Chlorella CGF extract, 1-4 parts by weight of Spirulina phycocyanin, and 1-3 parts by weight of Haematococcus pluvialis astaxanthin microcapsules; preferably, the mass ratio of Chlorella CGF extract, Spirulina phycocyanin, and Haematococcus pluvialis astaxanthin microcapsules is 1:1:

1.

3. The composition according to claim 1, characterized in that, The Chlorella is Chlorella proteolyticus, and the Spirulina is Spirulina platensis.

4. The composition according to claim 3, characterized in that, The total nucleotide and amino acid content of the Chlorella CGF extract is ≥35%; the purity of the Spirulina platensis phycocyanin is ≥25% or the color value is E. 18 ≥60; and / or the astaxanthin content in the Haematococcus pluvialis astaxanthin microcapsules is ≥2.0%.

5. The composition according to claim 4, characterized in that, The Chlorella proteoglycans CGF extract is obtained by steam-heat swelling and cell wall disruption, hot water extraction, ultrafiltration, concentration, and drying of Chlorella proteoglycans; the Haematococcus pluvialis astaxanthin microcapsules are obtained by the following method: Haematococcus pluvialis is subjected to cell wall disruption treatment, then mixed with wall material, emulsified, and finally made into powdered microcapsules by spray drying; or the Spirulina platensis phycocyanin is obtained by including Spirulina platensis powder extraction, centrifugation of the extract, purification of the centrifugation supernatant, and drying.

6. The composition according to any one of claims 1-5, characterized in that, The mass ratio of the compound microalgae active ingredients, taurine, and vitamin C is (3-15):(1.5-4.5):(0.5-1); Preferably, the mass ratio of the compound microalgae active ingredient, taurine, and vitamin C is (3-9):(2-4):

1.

7. A solution with anti-fatigue steady-state function, characterized in that, The composition comprising any one of claims 1-6, wherein the mass percentage of the composite microalgae active ingredient is 0.25-2.2% by mass of the solution.

8. The solution according to claim 7, characterized in that, It also includes one or more of the following: sweeteners, acidulants, stabilizers, color stabilizers, and food flavorings; Preferably, the sweetener accounts for 2-7% of the solution mass; the sweetener is selected from one or more of xylitol, lactitol, sucrose, sucralose, glucose, fructose, erythritol, maltitol, steviol glycosides, and mogrosides. Preferably, the acidulant accounts for 0.12-0.6% of the solution mass; the acidulant is selected from one or more of citric acid, L-malic acid, phosphoric acid, lactic acid, acetic acid, sodium citrate, sodium dihydrogen phosphate, and sodium hydrogen phosphate. Preferably, the stabilizer accounts for 0.06-0.35% of the solution mass; the stabilizer is selected from one or more of pectin, gellan gum, xanthan gum, sodium carboxymethyl cellulose, carrageenan, and agar. Preferably, the color-protecting stabilizer accounts for 0.03-0.18% of the solution mass; the color-protecting stabilizer is selected from one or more of L-sodium ascorbate, sodium phytate, disodium ethylenediaminetetraacetate, and D-isoascorbate. Preferably, the edible flavoring accounts for 0.05-0.25% of the total mass of the solution.

9. A method for preparing the solution according to any one of claims 6-8, characterized in that, Includes the following steps: (1) Preparation of compound microalgae active ingredients: The CGF extract of Chlorella pulvinata and phycocyanin of Spirulina platensis were added to a portion of room temperature pure water and stirred until completely dissolved. The mixture was filtered through a microporous membrane to remove impurities and obtain water-soluble microalgae active liquid. Astaxanthin microcapsules of Haematococcus pluvialis were added to water and stirred at low speed to disperse evenly to obtain astaxanthin microcapsule suspension. (2) Preparation of colloidal solution: The stabilizer and sweetener are pre-dry mixed evenly, added to water, stirred at high speed until completely swollen and dissolved, cooled to obtain colloidal solution; (3) Nutritional preparation: Add acidulant, color stabilizer and taurine to colloidal solution in sequence and stir until completely dissolved; then slowly add water-soluble microalgae active liquid and astaxanthin microcapsule suspension, stir evenly and then add vitamin C and edible flavoring, add purified water to make up the volume to obtain the liquid. (4) Homogenization and degassing: The liquid feed is homogenized and degassed under high pressure; (5) Instant sterilization.

10. The use of a composition according to any one of claims 1-6 or a solution according to any one of claims 7-8 in the preparation of antioxidant and anti-fatigue products.

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