Novel anti-fatigue coffee solid beverage and preparation method thereof

Abstract

The invention discloses a novel anti-fatigue coffee solid beverage and a preparation method thereof. The chlamydomonas reinhardtii protein peptide matcha milk is prepared from the following components in parts by mass: 8-16 parts of coffee-containing extraction concentrated solution, 3-7 parts of chlamydomonas reinhardtii protein peptide, 1-5 parts of matcha powder, 50-90 parts of condensed fresh milk and 1-5 parts of cane sugar. The ABTS clearance rate and the comprehensive sensory score are used as evaluation indexes, and the production formula of the solid beverage is optimized by adopting a method of optimizing a BP neural network by adopting a grey wolf algorithm. The coffee solid beverage contains novel edible green alga protein peptide-chlamydomonas reinhardtii protein peptide, has remarkable ABTS free radical scavenging activity and in-vivo anti-fatigue activity, and can construct a new form of health food with a synergistic anti-oxidation function with caffeine and tea polyphenol.

Description

Technical Field

[0001] This invention belongs to the field of functional food technology, specifically relating to a novel anti-fatigue coffee solid beverage and its preparation method. Background Technology

[0002] With the global prevalence of chronic fatigue continuing to rise, fatigue has evolved into a major public health problem. Persistent fatigue not only reduces quality of life but is also closely related to inflammation, aging, and nervous system dysfunction. Current technology classifies exercise fatigue into two main types: peripheral fatigue and central fatigue. The former is located in musculoskeletal muscles and other motor organs, characterized by energy metabolism imbalance and decreased muscle output; the latter originates from central nervous system dysfunction, manifested as abnormal neurotransmitter synaptic transmission and cognitive and emotional disorders (Bigland-Ritchie B., Woods JJ Changes in muscle contractile properties and neural control during human muscular fatigue[J].Muscle and Nerve,1984,7(9).). Studies have confirmed that high-intensity exercise triggers oxidative stress, disrupting the free radical / reactive oxygen species balance. Excessive ROS accumulation is the core factor leading to fatigue (Nie Jinlei, Zhang Yong. New progress in exercise and free radical research [J]. Journal of Tianjin University of Sport, 2001, 16(2):32-34.; Li Yongchun. Free radicals and athletic ability [J]. Sports World, 2008, 8:104-105.). Although the body possesses non-enzymatic and enzyme-based antioxidant defense networks, these are insufficient to eliminate free radicals under continuous exercise load. Therefore, developing a novel anti-fatigue composition that can simultaneously regulate peripheral energy metabolism, improve central neurotransmitter transmission, and enhance antioxidant defense has become a critical technological bottleneck that urgently needs to be overcome in this field.

[0003] Chlamydomonas reinhardtii, a high-protein microalgae, has shown potential bioactivity in its protein extracts in in vitro studies, demonstrating its development value as a functional food ingredient (Liu Meiting, Mao Ya, Xie Jintao, et al. Extraction of Chlamydomonas reinhardtii protein and screening of monoamine oxidase A inhibitory peptides [J / OL]. Food Industry Technology, 2025). However, existing commercially available Chlamydomonas reinhardtii products are mainly in single forms such as meal replacement powders and tablets, and there are no solid beverages that synergistically combine it with functional factors such as caffeine and tea polyphenols. The high-value utilization pathways of microalgae protein remain relatively limited. Solid beverages have become an important dosage form for ready-to-eat nutritional supplements due to their convenience, quick preparation, and adjustable formulation. However, how to achieve synergistic effects of functional components while ensuring sensory acceptability remains a key challenge facing the industry.

[0004] In recent years, the artificial neural network-gray wolf algorithm coupling technology has shown high accuracy and fast convergence advantages in processing multi-component-multi-objective optimization of food. It can simultaneously incorporate sensory, physicochemical and efficacy indicators into the model to achieve intelligent optimization of formula (Li Z., Tam V., Yeung L. An adaptive multi-population optimization algorithm for global continuous optimization[J].IEEE Access,2021.).

[0005] Therefore, developing a ternary composite solid beverage of Chlamydomonas reinhardtii-matcha-coffee based on the gray wolf algorithm-optimized BP neural network can not only fill the technological gap of microalgal protein peptides in the field of high antioxidant solid beverages, but also provide a new path for the intelligent manufacturing of functional foods. Summary of the Invention

[0006] To address the aforementioned problems, the present invention aims to provide a coffee solid beverage that helps combat fatigue. The production formula of the solid beverage was optimized using the ABTS clearance rate and overall sensory score as evaluation indicators, and a backpropagation neural network optimized by the gray wolf algorithm.

[0007] The above-mentioned objective of this invention is achieved through the following technical solution:

[0008] This invention provides a novel anti-fatigue coffee solid beverage, characterized by comprising the following components: coffee extract concentrate, Chlamydomonas reinhardtii protein peptide PAP, matcha, fresh milk condensed milk, and sucrose.

[0009] The present invention also provides a method for preparing the above-mentioned novel anti-fatigue coffee solid beverage, characterized by comprising the following steps:

[0010] (1) Weigh an appropriate amount of coffee powder, add water in a certain proportion to extract coffee extract concentrate.

[0011] (2) Add coffee extract concentrate, Chlamydomonas rhizogenes protein peptide PAP, matcha powder, fresh milk condensed milk and sucrose syrup to the concentrate in proportion and mix them.

[0012] (3) Homogenize the prepared concentrate using a high-speed dispersion homogenizer;

[0013] (4) The preparation solution is pre-frozen and then freeze-dried to obtain freeze-dried coffee solid beverage powder;

[0014] (5) Sterilize the freeze-dried powder using a pasteurizer;

[0015] (6) Determine the ABTS clearance rate of coffee solid beverages and conduct sensory evaluation on them;

[0016] (7) Vacuum-packed and stored at low temperature;

[0017] (8) Input the obtained single-factor data into the BP neural network, use the gray wolf algorithm to find the best production formula, and verify it.

[0018] Further, in step (1), the ratio of coffee powder to water is 1:2 (mass-volume ratio, g / mL) for extraction.

[0019] Further, in step (2), the ingredients are prepared according to the mass proportions, including 8-16 parts of coffee extract concentrate, 3-7 parts of Chlamydomonas reinhardtii protein peptide, 1-5 parts of matcha powder, 50-90 parts of fresh milk condensed milk, and 1-5 parts of sucrose.

[0020] Furthermore, in step (2), the preparation process of Chlamydomonas reinhardtii protein peptide PAP is as follows:

[0021] (1) Extraction of proteins from Chlamydomonas reinhardtii (alkali dissolution and acid precipitation method)

[0022] ① Mix Chlamydomonas reinhardtii algal powder with DES reagent containing 72.23% water (mass fraction) at a ratio of 1:10 (g / mL) until homogeneous;

[0023] ②Alkali dissolution: Adjust the pH to about 9.5 with NaOH, and after low-temperature swelling and ultrasonic treatment, collect the supernatant by centrifugation;

[0024] ③ Acid precipitation: Add 1 volume of 60% (mass fraction) ammonium sulfate solution and 2 volumes of saturated sodium sulfate solution to the supernatant, mix well, adjust the pH to about 3.5, let stand at low temperature, centrifuge to collect the precipitate, redissolve in ultrapure water, dialyze, and freeze dry to obtain Chlamydomonas reinhardtii protein.

[0025] (2) Take Chlamydomonas reinhardtii protein, prepare a protein solution with ultrapure water, mix in a water bath and adjust the pH to about 6.0, add an appropriate amount of papain, and enzymatically hydrolyze at about 55°C, keeping the pH stable during the process. After inactivation in a boiling water bath, freeze dry to obtain Chlamydomonas reinhardtii protein peptide PAP.

[0026] Furthermore, the DES reagent consists of choline chloride and glycerol in a volume ratio of 1:1.

[0027] Furthermore, the low temperature in the acid precipitation step is 4°C.

[0028] Furthermore, the concentration of the Chlamydomonas reinhardtii protein solution was 5 mg / mL; the enzymatic hydrolysis time was 4 h.

[0029] Furthermore, the Chlamydomonas reinhardtii protein peptide PAP was purified to obtain three ultrafiltration fractions: PAP-I (molecular weight >10k), PAP-II (molecular weight <10k), and PAP-III (molecular weight <3k). The fraction with the strongest ABTS free radical scavenging ability was selected for peptide sequence identification and screening.

[0030] Furthermore, the anti-fatigue effect of Chlamydomonas reinhardtii protein peptide PAP in mice is mainly manifested in its ability to alleviate peripheral fatigue and central nervous system fatigue.

[0031] Furthermore, Chlamydomonas reinhardtii protein peptides (PAPs) achieve their anti-fatigue effects by regulating peripheral energy metabolism, improving central nervous system function, and scavenging excess free radicals.

[0032] Furthermore, in step (3), the high-speed dispersion homogenizer is used to homogenize for 60 seconds at 8000 rpm.

[0033] Furthermore, in step (5), the pasteurizer is used to sterilize the food at 75°C for 25 seconds.

[0034] Furthermore, in step (6), during the sensory evaluation, each packet of freeze-dried powder is mixed with water at a ratio of 1:20 (mass-volume ratio, g / mL).

[0035] Furthermore, in step (7), the low-temperature storage temperature is 0-4℃.

[0036] Further, in step (8), the optimal production formula obtained by screening is, by mass parts: 11.9 parts coffee extract concentrate, 4 parts Chlamydomonas reinhardtii protein peptide, 2.1 parts matcha powder, 80 parts fresh milk condensed milk, and 3 parts sucrose.

[0037] The advantage of this invention lies in its intelligent formulation design for a ternary solid beverage combining Chlamydomonas reinhardtii, matcha, and coffee by combining the Grey Wolf optimization algorithm with a backpropagation neural network. This not only enhances the product's functionality and stability but also expands the application avenues for microalgae proteins in high-value-added foods. This method can simultaneously optimize multiple key indicators, including sensory evaluation, physicochemical properties, antioxidant activity, and anti-fatigue effects, thereby significantly enhancing the product's functionality while ensuring its taste and flavor. This provides a new technological path for the intelligent manufacturing of functional foods.

[0038] The beneficial effects of this invention lie in the significant advantages that the newly developed anti-fatigue coffee compound solid beverage exhibits in anti-fatigue, antioxidant, and neuroprotective properties. It effectively regulates peripheral energy metabolism, improves central nervous system function, and eliminates excess free radicals, thereby alleviating the various negative effects of exercise fatigue. Furthermore, the product is convenient to carry, quick to prepare, and has flexible formulations, facilitating market promotion and consumer acceptance, and contributing to the popularization and upgrading of functional foods. Attached Figure Description

[0039] Figure 1 The effect of coffee extract concentrate addition on ABTS clearance rate and sensory score of coffee solid beverage.

[0040] Figure 2 The effect of the amount of Chlamydomonas chinensis protein peptide added on ABTS clearance rate and sensory score of coffee solid beverage.

[0041] Figure 3 The effect of matcha addition on ABTS clearance rate and sensory score of coffee solid beverages.

[0042] Figure 4 The effect of the amount of fresh milk and condensed milk added on ABTS clearance rate and sensory score of coffee solid beverages.

[0043] Figure 5 The effect of sucrose addition on ABTS clearance rate and sensory score of coffee solid beverages.

[0044] Figure 6 GPC gel permeation chromatograms of each ultrafiltration component of PAP ( Figure 6 a: PAP-I; Figure 6 b: PAP-II; Figure 6 c: PAP-III).

[0045] Figure 7 IC50 of the ABTS radical scavenging capacity of the four PAP ultrafiltration components (different lowercase letters in the figure indicate significant differences in the ABTS radical scavenging capacity of the four components (p<0.05)).

[0046] Figure 8 This is the total ion chromatogram.

[0047] Figure 9 The effect of PAP on forelimb gripping force in fatigue model mice (NC, blank group; M, model group; P, positive group; L, low-dose group; H, high-dose group).

[0048] Figure 10 The effect of PAP on exhaustive swimming time in fatigue model mice (NC, blank group; M, model group; P, positive group; L, low-dose group; H, high-dose group).

[0049] Figure 11 PAP was used to treat blood urea nitrogen (BUN) in fatigue model mice. Figure 11 a) and lactate dehydrogenase LDH ( Figure 11 b) Effect of concentration (NC, blank group; M, model group; P, positive group; L, low-dose group; H, high-dose group).

[0050] Figure 12 PAP-induced effects on liver glycogen LG in fatigue model mice Figure 12 a) and muscle glycogen MG ( Figure 12 Effect of b) (NC, blank group; M, model group; P, positive group; L, low-dose group; H, high-dose group).

[0051] Figure 13 PAP is used to treat malondialdehyde (MDA) in fatigue model mice. Figure 13 a) Superoxide dismutase (SOD) Figure 13 b) and GSH-px ( Figure 13 c) Effect of levels (NC, blank group; M, model group; P, positive group; L, low-dose group; H, high-dose group).

[0052] Figure 14 HE staining of gastrocnemius muscle in fatigue model mice (×200).

[0053] Figure 15 PAP is used to treat 5-hydroxytryptamine (5-HT) in fatigue model mice. Figure 15 a) Dopamine (DA) Figure 15 b) and norepinephrine (NE) Figure 15 Effects of c) (NC, blank group; M, model group; P, positive group; L, low-dose group; H, high-dose group).

[0054] Figure 16 The effect of PAP on open field experiments in fatigue model mice ( Figure 16 a: Number of squares spanned; Figure 16 b: Number of times the object stood upright; Figure 16 c: number of cheek-grabbing sessions (NC, blank group; M, model group; P, positive group; L, low-dose group; H, high-dose group).

[0055] Figure 17 The effect of PAP on tail suspension time in fatigue model mice (NC, blank group; M, model group; P, positive group; L, low-dose group; H, high-dose group).

[0056] Figure 18 HE staining of the hippocampus of fatigue model mice (200x magnification).

[0057] Figure 19 Pearson correlation analysis of antioxidant and anti-fatigue effects in fatigue model mice.

[0058] Figure 20 This is a flowchart illustrating the preparation process of a novel anti-fatigue coffee solid beverage. Detailed Implementation

[0059] The present invention will be further described below with reference to the accompanying drawings and specific embodiments, but the embodiments do not limit the present invention in any way. Unless otherwise specified, the reagents, methods and equipment used in the present invention are conventional reagents, methods and equipment in this technical field.

[0060] Unless otherwise specified, all reagents and materials used in the following examples are commercially available.

[0061] The Chlamydomonas reinhardtii protein peptides used in this invention were prepared in the laboratory, and the Chlamydomonas reinhardtii was purchased from Shanxi Touyun Biotechnology Co., Ltd. All solvents used in the experiment were of analytical grade.

[0062] Example 1

[0063] This embodiment provides a single-factor optimization experiment for a novel anti-fatigue coffee solid beverage formulation:

[0064] The preparation method includes the following steps:

[0065] (1) Extraction of proteins from Chlamydomonas reinhardtii (alkali dissolution and acid precipitation method)

[0066] ① Mix Chlamydomonas reinhardtii algal powder with DES reagent (choline chloride: glycerol = 1:1, v / v) with a water content of 72.23% (mass fraction) at a material-to-liquid ratio of 1:10 (g / mL).

[0067] ②Alkali dissolution: Adjust the pH to about 9.5 with NaOH, swell at 4℃ for 5 hours, sonicate for 11 minutes (557w, room temperature), and collect the supernatant by centrifugation.

[0068] ③ Acid precipitation: Add 1 volume of 60% (mass fraction) ammonium sulfate solution and 2 volumes of saturated sodium sulfate solution to the supernatant, mix well, adjust the pH to about 3.5, let stand at 4℃ for 18 hours, centrifuge to collect the precipitate, redissolve in ultrapure water and dialyze, and freeze dry to obtain Chlamydomonas reinhardtii protein.

[0069] (2) Take Chlamydomonas reinhardtii protein, prepare a protein solution of 5 mg / mL with ultrapure water, mix in a water bath and adjust the pH to about 6.0, add an appropriate amount of papain, and enzymatically hydrolyze at about 55℃ for 4 hours, keeping the pH stable during the process. After inactivation in a boiling water bath, freeze dry to obtain Chlamydomonas reinhardtii protein peptide PAP.

[0070] (3) Preparation of coffee extract concentrate: Weigh 18g of coffee powder, add water at a ratio of 1:2 (mass-volume ratio, g / mL) to extract coffee extract concentrate.

[0071] (4) Coffee extract concentrate, Chlamydomonas reinhardtii peptides, matcha powder, fresh milk condensed milk, and sucrose syrup were compounded in a certain proportion. The fixed amounts of the added components were: 70g fresh milk condensed milk, 4g Chlamydomonas reinhardtii peptides, 3g matcha powder, and 2g sucrose. Based on this, the addition gradients of coffee extract concentrate were set at 8g, 10g, 12g, 14g, and 16g to investigate the effects of the amount of coffee extract concentrate added on the ABTS free radical scavenging rate and sensory score of coffee solid beverage.

[0072] (5) The prepared concentrate was homogenized using a high-speed dispersion homogenizer (8000 rpm, 60 s). The homogenized liquid was pre-frozen at -20℃ for 24 h and then freeze-dried to obtain freeze-dried coffee solid beverage powder. The freeze-dried powder was sterilized using a pasteurizer (75℃, 25 s). The ABTS removal rate of the coffee solid beverage was determined and its sensory evaluation was performed. Finally, it was vacuum-packed and stored at 0-4℃.

[0073] Example 2

[0074] The difference from Example 1 is that the amount of fixed components added in step (4) is: 10g of coffee concentrate, 70g of fresh condensed milk, 3g of matcha powder, and 2g of sucrose. Based on this, the addition gradient of Chlamydomonas reinhardtii protein peptides was set at 3g, 4g, 5g, 6g, and 7g to investigate the effect of the amount of Chlamydomonas reinhardtii protein peptides added on the ABTS free radical scavenging rate and sensory score of coffee solid beverage.

[0075] Example 3

[0076] The difference from Example 1 is that the amount of fixed components added in step (4) is: 10g of coffee concentrate, 5g of Chlamydomonas reinhardtii peptide, 70g of fresh milk condensed milk, and 2g of sucrose. Based on this, the addition gradient of matcha powder was set to 1g, 2g, 3g, 4g, and 5g to investigate the effect of the amount of matcha powder added on the ABTS free radical scavenging rate and sensory score of coffee solid beverage.

[0077] Example 4

[0078] The difference from Example 1 is that the amount of fixed components added in step (4) is: 10g of coffee concentrate, 5g of Chlamydomonas reinhardtii peptide, 2g of matcha powder, and 2g of sucrose. Based on this, the addition gradient of fresh milk and condensed milk was set at 50g, 60g, 70g, 80g, and 90g to investigate the effect of the amount of fresh milk and condensed milk added on the ABTS free radical scavenging rate and sensory score of coffee solid beverage.

[0079] Example 5

[0080] The difference from Example 1 is that the amount of fixed components added in step (4) is: 10g of coffee concentrate, 5g of Chlamydomonas reinhardtii peptide, 2g of matcha powder, and 80g of fresh condensed milk. Based on this, the addition gradient of sucrose is set at 1g, 2g, 3g, 4g, and 5g to explore the effect of sucrose addition on the ABTS free radical scavenging rate and sensory score of coffee solid beverage.

[0081] Example 6: Artificial Neural Network Algorithm Optimization Experiment

[0082] Includes the following steps

[0083] (1) Establishment and training of the BP neural network: A neural network model was constructed in the Matlab R2022b environment. The network structure was set as follows: the transfer function of the hidden layer was tansig, the output layer was purelin, and the training algorithm was Levenberg-Marquardt. After normalization, the experimental data were divided into training set, test set and simulation set according to random seeds. The input variables, as shown in Table 1, included the amount of coffee concentrate, Chlamydomonas reinhardtii protein peptide and matcha added. The output variables were sensory score and ABTS free radical scavenging rate. The training data came from the results of single-factor experiments.

[0084] Table 1 Factor Level Coding Table

[0085]

[0086] (2) Gray Wolf Algorithm Optimization Experiment: Combining the gray wolf optimization algorithm with a backpropagation (BP) neural network can effectively optimize the weights and thresholds of the neural network, thereby improving the network performance and prediction accuracy, and ultimately outputting better results. The specific steps are as follows: ① Initialize the gray wolf population and randomly generate initial positions and velocities → ② Calculate the gray wolf fitness values ​​and determine the positions of gray wolves such as Alpha, Beta, and Delta → ③ Update the weights and thresholds according to the positions of gray wolves such as Alpha, Beta, and Delta, and calculate the new fitness values ​​→ ④ Repeat steps ② and ③ until the stopping condition is met → ⑤ Output the optimized weights and thresholds to obtain the optimized neural network model. Among them, the population size is set to 20 and the number of iterations is 50.

[0087] (3) Verify and output the optimal model of the Grey Wolf optimization algorithm: The trained and parameter-optimized neural network model is fixed and saved using the sim function. The optimal network parameters of this model are used to achieve the final prediction of the target indicators (sensory score and ABTS free radical scavenging rate). Using R... 2 Using MAPE as the screening index, the network model with the best sensory score and ABTS removal rate was selected, and its predicted optimal process was experimentally verified.

[0088] Isolation and purification of 1PAP

[0089] 1.1 Ultrafiltration purification of PAP

[0090] Ultrafiltration was performed using an ultrafiltration cup to obtain Chlamydomonas reinhardtii protein peptide fractions of different molecular weights. Under nitrogen pressure (0.2 MPa), the protein peptide solution was sequentially passed through ultrafiltration membranes with molecular weight cutoffs of 10 kDa and 3 kDa to achieve molecular weight fractionation. The ABTS free radical scavenging capacity of each fraction was determined. The separated fractions included: fractions with PAP greater than 10 kDa (PAP-I), fractions with PAP between 3 kDa and 10 kDa (PAP-II), and fractions with PAP less than 3 kDa (PAP-III). The molecular weight of each fraction was determined using a PL-GPC120.

[0091] 1.2 Peptide Sequence Identification and Screening for PAP-III

[0092] The amino acid sequences of peptides in the PAP fraction (PAP-III) were identified using LC-MS / MS. Mobile phase A was a 0.1% (v / v) formic acid solution, and mobile phase B was a 0.1% (v / v) formic acid acetonitrile-water mixture (acetonitrile v / v 84%). Chromatographic separation was performed using a reversed-phase C18 column (0.15 mm with reversed-phase fractional mobile phase). The sample was automatically injected into a Zorbax 300SB-C18 peptide delivery tube for separation. Mass spectrometry detection was performed using electrospray ionization positive ion mode (ESI+), with a scan range of 300–1800 m / z.

[0093] Peptide score (P-Score) is a key indicator in mass spectrometry data analysis, used to assess the reliability of peptide identification results. It uses an algorithm to score the degree of matching between the peptide and the experimental mass spectrum; a higher score indicates a higher degree of matching and a more reliable identification result. Generally, a P-Score > 100 indicates high reliability. The Peptide Ranker Responses (PRR) system is a bioactive peptide prediction tool based on a neural network algorithm. This server uses a computational model to evaluate the bioactivity of user-submitted peptide sequences, outputting a probability value between 0 and 1. According to industry-standard criteria, a PR greater than 0.5 indicates that the peptide has potential bioactivity. Therefore, peptide sequences are screened based on a P-Score > 100 and a PRR > 0.5 for further research.

[0094] Based on peptide information obtained by LC-MS / MS, the molecular structures were obtained and plotted using the Public Chemical Database (PubChem) and ChemDraw 19.0 software. "Drug-likeness" characteristics were identified based on the SwissADME database. SwissADME is a research tool used to predict the pharmacokinetic (i.e., absorption, distribution, metabolism, and excretion) properties, physicochemical properties, drug-likeness, and medicinal chemical friendliness of small molecules. Developed by the Swiss Institute for Bioinformatics, it aims to rapidly evaluate the potential performance of candidate compounds in the early stages of drug development. According to the Lipinski rule evaluation results of the SwissADME platform, when a peptide receives a "good drug-likeness" evaluation result, it indicates that it possesses the following characteristics: good drug-likeness and potential biological activity. Therefore, this platform can be used to search for potentially active peptides with fatigue-relieving functions.

[0095] 2. Determination of ABTS free radical scavenging ability: Mix 1 mL of ABTS stock solution (7 mmol / L) with 88 μL of K2S2O8 solution (140 mmol / L), react in the dark for 12-16 h, then dilute with 95% (v / v) ethanol at 734 nm to an absorbance of 0.70 ± 0.02, which serves as the ABTS working solution. Take 50 μL of the sample solution and mix with 150 μL of ABTS dilution solution, shake well at room temperature, let stand for 10 min, and measure the absorbance OD. 734 .

[0096] ABTS clearance rate = 1 - A S / A0(Equation 1), where A S —Sample absorbance; A0 —Ultrapure water instead of sample absorbance.

[0097] 3. Sensory Evaluation Method: Ten evaluators with professional sensory analysis training conducted a sensory evaluation of the Chlamydomonas reinhardtii matcha compound solid beverage. Each 18g packet of freeze-dried powder was reconstituted with water at a ratio of 1:20 (mass-volume ratio, g / mL), and sensory scores were then calculated. The evaluation score was out of 100, and the specific scoring criteria are shown in Table 2.

[0098] Table 2 Sensory Evaluation Criteria

[0099]

[0100]

[0101] 4. Determination of the anti-fatigue activity of Chlamydomonas reinhardtii protein peptide PAP in mice

[0102] 4.1 Experimental animals: 53 SPF-grade ICR male mice aged 6 - 8 weeks were used, with the license number: SCXK(Guangdong)2020 - 0051, and the initial body weight was 18 - 22 g. The experimental animals were acclimated under standard conditions (temperature 20 - 26°C, humidity 40% - 70%) for 7 days. During the acclimation period, the mice drank water and ate normally, and the breeding environment was strictly disinfected and kept clean.

[0103] 4.2 Model establishment: A fatigue model was established using the chronic multi-factor composite stress stimulation method (chronic restraint + chronic unpredictable mild stress + excessive exercise), simulating pathogenic factors such as modern human overloaded operation, etc., to replicate irregular factors in all aspects of humans to the greatest extent and induce fatigue symptoms. The experiment was modeled for 28 days, and two stimulation methods were selected every day, mainly including: chronic restraint for 5 h, fasting and water deprivation for 24 h, wet bedding for 24 h, forced swimming for 10 min, running on a rotating wheel for 15 min, shaking the mouse cage for 5 min, crowding for 24 h, and circadian rhythm inversion.

[0104] 4.3 Grouping and administration: Mice in different groups were gavaged according to Table 3 for 28 days. The fatigue model was established using the modeling method in 4.2, and the experiment was carried out with the method of administering drugs while modeling.

[0105] Table 3 Grouping and gavage conditions of mice

[0106]

[0107] 4.4 Behavioral experiments: Experiments were carried out before and after modeling (day 0 and day 28).

[0108] 4.4.1 Load-bearing swimming experiment: The load-bearing swimming experiment is an effective tool for studying exercise-induced fatigue by simulating high-intensity exercise, evaluating exercise endurance and fatigue degree. A lead sheet about 5% of the body weight was wound around the root of the mouse's tail, and the mouse was placed in a constant-temperature water bath (water temperature 25°C, kept at a constant temperature, water depth ≥, continuously stirring the water surface to ensure that the mouse was always in a swimming state). Timing started. When the mouse's head was submerged in the water for 10 s, the mouse was considered to have reached the exhaustion state, and this was the mouse's exhaustion swimming time.

[0109] 4.4.2 Forelimb grip strength experiment: The size of the forelimb grip strength is the most intuitive evaluation index for judging exercise-induced fatigue. The smaller the strength, the more serious the fatigue state. The forelimb grip strength of the mouse was evaluated by the grip strength. The mouse was placed on a wire mesh, and the mouse's tail was pulled backward horizontally until its front paws completely left the wire mesh.

[0110] 4.4.3 Open field experiment: By recording the spontaneous activity of the mouse in an open area, the degree of central nervous fatigue was judged. The mouse was placed in a black box with 25 grids (50 cm in the open area), and the movement of the mouse within 5 min was recorded.

[0111] 4.4.4 Tail Suspension Test: Central nervous system fatigue is often accompanied by behavioral despair. The tail suspension test can be used to observe the struggling and immobility time of mice, assess fatigue status, and reflect the excitation and inhibition balance of the central nervous system. The mouse's tail is suspended on an iron stand with its nose tip approximately 25 cm above the horizontal plane. The mouse's intermittent immobility and fatigue state are observed. Each mouse is tested for 6 minutes, including a 1-minute adaptation period and a 5-minute experimental period. The immobility time in the last 5 minutes is recorded and analyzed.

[0112] 4.4.5 Collection of Mouse Blood and Organs: After the animal model was established, mice were euthanized by intraperitoneal anesthesia and dissected. The specific procedures were as follows: First, blood samples were collected: whole blood was collected through the eyeball into 2 mL centrifuge tubes. After clotting for 1 hour, the blood was centrifuged at 3000 r / min for 10 min to separate the serum, aliquoted, and cryopreserved at -80°C. Next, tissue samples were collected: whole brain, skeletal muscle, heart, liver, and kidney tissues were harvested in sequence. Some tissues were aliquoted and stored at -80°C for subsequent experimental analysis.

[0113] 4.4.6 Determination of mouse muscle tissue index: After dissecting the mouse muscle tissue, clean it with physiological saline, dry it, and weigh it.

[0114] Muscle tissue index (%) = m1 / m0 × m tissue (Equation 2), where m1 represents the weight of the left and right legs of the mouse, g; and m0 represents the body weight of the mouse, g.

[0115] 4.4.7 Histopathology HE (hematoxylin-eosin staining) sections: Mouse hippocampal and muscle tissues were stained with HE and then subjected to histopathological observation to evaluate the structural changes under optical microscopy.

[0116] 4.4.8 ELISA Detection and Biochemical Indicator Determination: After the last administration of the drug to establish the mouse model, blood was collected from living mice and tissues were dissected. ELISA and biochemical indicators were then measured. The levels of blood urea nitrogen (BUN) in serum, liver glycogen (LG), and muscle glycogen (MG) in liver and muscle tissues were detected using biochemical kits. The levels of serotonin (5-HT), dopamine (DA), and norepinephrine (NE) in brain tissue, and the activities of MDA, superoxide dismutase (SOD), glutathione peroxidase (GSH-px), and serum lactate dehydrogenase (LDH) in liver tissue were detected using ELISA kits.

[0117] 5. Statistical Analysis: Experimental data were statistically analyzed using GraphPad Prism 9 software. Statistical differences were represented by the following notations: Blank group vs. model group: #p<0.05, ##p<0.01, ###p<0.001, ####p<0.0001; Model group vs. dose group: *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001.

[0118] 6. Experimental Results

[0119] 6.1 Results of Single-Factor Experiment

[0120] 6.1.1 Effect of coffee concentrate addition amount on ABTS free radical scavenging rate and sensory properties of coffee solid beverages

[0121] All other conditions being equal, by Figure 1 The results of the α-radical scavenging rate showed that as the amount of coffee concentrate added increased, the sensory score first increased and then decreased, while ABTS scavenging initially increased and then stabilized. The sensory score was optimal when the amount of coffee concentrate added was 10g, with the ABTS scavenging rate stabilizing at 74.67%. At this level, the beverage was rich in coffee aroma, had a full and smooth taste, and exhibited high antioxidant activity. When the amount of coffee concentrate added exceeded 12g, although there was no significant change in antioxidant activity, the coffee flavor became too strong, the taste slightly bitter, and the aroma of milk and tea powder was partially masked, resulting in a lower sensory score. The effect of the amount of coffee concentrate added on the sensory characteristics of the beverage is as follows: Figure 1 As shown in b, with the addition of coffee concentrate, the changes in texture, color, aroma, taste, and solubility all initially increase and then decrease, reaching their maximum values ​​at an addition amount of 10g. Therefore, the optimal amount of coffee concentrate to add is 10g.

[0122] 6.1.2 Effects of Chlamydomonas reinhardtii protein peptide addition on ABTS free radical scavenging rate and sensory properties of coffee solid beverages

[0123] All other conditions being equal, by Figure 2 The results of the free radical scavenging rate show that as the amount of Chlamydomonas reinhardtii protein peptide added increases, the sensory score first increases and then decreases, while ABTS scavenging first increases and then tends to stabilize. When the amount of Chlamydomonas reinhardtii protein peptide added is 5g, the sensory score is optimal, and the ABTS scavenging rate stabilizes at 77.84%, which is the best addition amount. At this point, the solid beverage has the best taste. As the amount of Chlamydomonas reinhardtii protein peptide added increases further, its fishy smell becomes more pronounced, affecting the taste of the beverage and lowering the sensory score. The effect of the amount of Chlamydomonas reinhardtii protein peptide added on the sensory characteristics of the beverage is as follows: Figure 2As shown in b, with the addition of Chlamydomonas reinhardtii protein peptides, the changes in texture, color, odor, taste, and solubility all showed a trend of first increasing and then decreasing, reaching their maximum values ​​at an addition amount of 5g. In conclusion, the optimal addition amount of Chlamydomonas reinhardtii protein peptides is 5g.

[0124] 6.1.3 Effect of Matcha Addition Amount on ABTS Free Radical Scavenging Rate and Sensory Properties of Coffee Solid Beverages

[0125] All other conditions being equal, by Figure 3 The alpha free radical scavenging results show that as the amount of matcha added increases, the sensory score first increases and then decreases, while ABTS scavenging initially increases and then stabilizes. The optimal sensory score is achieved when the amount of matcha added is 3g, with the ABTS scavenging rate stabilizing at 83.58%. At this level, the beverage has the best taste and a suitable flavor balance. As the amount of matcha added increases, the powdery flavor of the matcha masks some of the milk and coffee aromas, affecting the taste of the beverage and lowering the sensory score. The effect of matcha addition on the sensory characteristics of the beverage is as follows: Figure 3 As shown in b, with the addition of matcha, the changes in texture, color, aroma, taste, and solubility all initially increased and then decreased, reaching their maximum values ​​at an addition amount of 3g. In conclusion, the optimal amount of matcha to add is 3g.

[0126] 6.1.4 Effects of Fresh Milk and Condensed Milk Addition on ABTS Free Radical Scavenging Rate and Sensory Properties of Coffee Solid Beverages

[0127] All other conditions being equal, by Figure 4 The ABTS scavenging results showed that as the amount of fresh milk and condensed milk added increased, the sensory score first increased and then decreased, while the ABTS scavenging rate remained relatively stable without significant change. The sensory score was optimal when the amount of fresh milk and condensed milk added was 80g, with the ABTS scavenging rate stabilizing at 82.04%. At this level, the beverage was at its best, with a perfect blend of fresh milk, coffee, and tea powder, and no off-flavors. As the amount of fresh milk and condensed milk added increased, the milk flavor masked the coffee aroma, resulting in a weak coffee flavor, affecting the beverage's taste and lowering the sensory score. The effect of the amount of fresh milk and condensed milk added on the sensory characteristics of the beverage is as follows: Figure 4 As shown in b, with the addition of fresh milk and condensed milk, the changes in texture, color, aroma, taste, and solubility all initially increased and then decreased, reaching their maximum values ​​at an addition amount of 80g. In conclusion, the optimal amount of fresh milk and condensed milk to add is 80g.

[0128] 6.1.5 Effects of sucrose addition on ABTS free radical scavenging rate and sensory properties of coffee solid beverages

[0129] All other conditions being equal, by Figure 5The results of the α-radical scavenging rate showed that as the amount of sucrose added increased, the sensory score first increased and then decreased, while the ABTS scavenging rate showed no significant change and tended to stabilize. The sensory score was optimal when the amount of sucrose added was 3g, and the ABTS scavenging rate stabilized at an optimal level of 81.32%. At this point, the taste was also optimal. As the amount of sucrose added increased, the beverage became too sweet, affecting its taste and lowering the sensory score. The effect of sucrose addition on the sensory characteristics of the beverage is as follows: Figure 5 As shown in b, with the addition of sucrose, the changes in texture, color, aroma, taste, and solubility all initially increased and then decreased, reaching their maximum values ​​at an addition of 3g. In conclusion, the optimal amount of sucrose to add is 3g.

[0130] 6.2 Results of the Gray Wolf Algorithm Optimization Experiment

[0131] The comparison results between the predicted and actual values ​​of the Grey Wolf optimization algorithm are shown in Table 4. Table 4 shows that the optimal process conditions are group 57. Following this optimal process, freeze-dried coffee was prepared using 11.9g of coffee concentrate, 4g of Chlamydomonas reinhardtii protein peptides, 2.1g of matcha powder, 80g of fresh condensed milk, and 3g of sucrose. The results showed that the actual sensory score was 94 points, and the actual ABTS clearance rate was 91.54%, which is close to the predicted value.

[0132] Table 4 Comparison of predicted and actual values ​​using the Grey Wolf optimization algorithm

[0133]

[0134]

[0135] 6.3 Results of PAP separation and purification

[0136] 6.3.1 Molecular weight distribution of different ultrafiltration components

[0137] PAP in the crude enzymatic hydrolysate was separated and purified using 3 kDa and 10 kDa ultrafiltration membranes. The molecular weight distribution of the ultrafiltration fractions (PAP-I, PAP-II, and PAP-III) was determined. Results are as follows: Figure 6 As shown, Figure 6 a shows the molecular weight distribution of PAP-I, with peaks mainly distributed in the molecular weight >10kJ range. Figure 6 b, 6c show the molecular weight distribution of PAP-II and PAP-III, with peaks mainly distributed in the molecular weight <10k; and Figure 6 Compared to b, Figure 6 The peak in c is mainly distributed in the molecular weight <3k, indicating that ultrafiltration is a scientific and effective method for separation and purification. The separated and purified component can be further studied.

[0138] 6.3.2 ABTS radical scavenging rate of different ultrafiltration components

[0139] Figure 7 IC50 values ​​for the ABTS radical scavenging efficiency of three ultrafiltration components 50 As shown in the graph, IC increases with increasing molecular weight. 50 As the molecular weight increases, the ABTS radical scavenging ability decreases. Among them, the component with a molecular weight <3k (PAP-III) has the strongest ABTS radical scavenging ability, so this component was selected for further analysis.

[0140] 6.3.3 Identification and screening of peptide sequences in PAP-based components

[0141] In LC-MS / MS analysis, the peptide score, derived from MaxQuant software, is an indicator used to assess the quality of the match between mass spectrometry data and the theoretical peptide sequence. A score > 100 is generally considered a high match and high confidence level. Peptide Ranker is a machine learning-based tool used to predict the bioactivity potential of peptides. The PeptideRanker Score is a predicted score for peptide bioactivity, ranging from 0 to 1. A higher score indicates a greater likelihood of bioactivity. When the PeptideRanker Score > 0.5, it typically indicates that the peptide has high bioactivity potential and can be considered as a candidate molecule for in vitro or in vivo experimental verification of its bioactivity. Therefore, combining... Figure 8 Based on the criteria of peptide length, peptide score greater than 100, and Peptide Ranker Score greater than 0.5, two peptides, LLPFP and FLSL, were obtained.

[0142] Based on peptide information from proteolytic hydrolysates obtained by LC-MS / MS, their "drug-like" properties were identified using the SwissADME database. The Lipinski criteria of SwissADME were used for screening and evaluation. The results showed that the FLSL peptide (Phe-Leu-Ser-Leu) received a rating of "high," indicating good drug-like properties. Therefore, the FLSL peptide is a potential active peptide with fatigue-improving effects, and subsequent Chlamydomonas reinhardtii protein peptides used in in vivo mouse studies all contained this peptide.

[0143] 6.4 Effects of PAP on anti-fatigue activity in mice

[0144] 6.4.1 Relieving peripheral fatigue

[0145] (1) Mouse muscle tissue index: As shown in Table 6, compared with the blank group, the muscle tissue of the model group mice was significantly reduced (p<0.05), indicating that the model was successful and the muscle mass of fatigued mice was reduced. Compared with the model group, the muscle tissue of the Rhodiola rosea positive group and the PAP administration group mice was significantly increased (p<0.05), suggesting that PAP can promote muscle growth, increase muscle strength output, and improve exercise capacity.

[0146] Table 6. Mouse muscle tissue index (%)

[0147] Note: Different lowercase letters in the figure represent significant differences in muscle tissue index among different groups of mice. The same letter indicates no significant difference between groups, while different letters indicate significant differences (p<0.05).

[0148] (2) Mouse forelimb grip strength: by Figure 9 It was found that before modeling and drug administration, there were no significant differences in the forelimb grip strength of mice in all experimental groups (p>0.05), with an average level of 1.36±0.41N, meeting the requirement of experimental group balance. Twenty-eight days after modeling and drug administration, the forelimb grip strength of mice in each group was 1.91±0.74N, 1.15±0.3N, 2.82±0.42N, 2.11±0.61N, and 2.31±1.04N, respectively. Compared with the blank group, the forelimb grip strength of mice in the model group was reduced (p<0.05), while the positive group and the PAP-treated group showed significant increases (p<0.01). Therefore, the multi-factor induction method used in this experiment to construct a fatigue mouse model is reliable, and the effects of PAP and Rhodiola rosea are comparable, both significantly improving fatigue and exhibiting a certain dose-dependent characteristic, promoting muscle growth and improving forelimb grip strength in fatigued mice.

[0149] (3) Exhaustion swimming time in mice: The exhaustion swimming test is commonly used to evaluate the anti-fatigue activity of compounds; the longer the time, the more significant the anti-fatigue effect of the compound. Figure 10Before drug administration, the average exhaustive swimming time of mice in each group was 8.52±2.67 min, with no significant difference (p>0.05). Twenty-eight days after drug administration, the exhaustive swimming times of mice in each group were 12.7±1.96 min, 6.32±1.37 min, 11.79±2.43 min, 10.29±2.1 min, and 11.38±7.99 min, respectively. The exhaustive swimming time of mice in the model group was significantly reduced compared to the control group (p<0.0001), while it was significantly increased in both the positive control group and the PAP-treated group (p<0.01). These results indicate that the fatigue mouse model was successfully established in this experiment, and both PAP and Rhodiola rosea significantly improved fatigue and enhanced swimming endurance in fatigued mice, exhibiting a certain dose-dependent characteristic. This may, to some extent, promote muscle growth, improve athletic ability, and thus enhance the athletic performance of mice.

[0150] (4) Blood urea nitrogen (BUN) and lactate dehydrogenase (LDH) in mice after exercise: such as Figure 11 As shown, after 28 days of modeling and drug intervention, biochemical indicators revealed that, compared with the blank control group, the serum urea nitrogen and lactate dehydrogenase levels in the model group mice were significantly increased (p<0.0001), reaching 0.2±0.01 mg / mL and 40.14±1.21 ng / L, respectively. This result confirms the successful establishment of the fatigue mouse model. When the body engages in prolonged strenuous exercise, the energy provided by aerobic respiration is insufficient. At this time, the anaerobic respiration energy supply pathway is activated, promoting the reduction of pyruvate to lactate and increasing the synthesis of adenosine triphosphate to produce energy for the body. However, at the same time, a large amount of lactate accumulates, and the activity of lactate dehydrogenase increases accordingly. After intervention with Rhodiola rosea and PAP, the levels of blood urea nitrogen (high dose: 0.14±0.02 mg / mL; positive control group: 0.13±0.02 mg / mL) in the experimental group mice were significantly improved compared with the model group (p<0.01), and the levels of lactate dehydrogenase (low dose: 34.06±3.19 ng / L; high dose: 33.71±0.85 ng / L; positive control group: 32.94±2.32 ng / L) were also significantly improved compared with the model group (p<0.01), indicating that the two drugs had a significant regulatory effect on the metabolic indicators of fatigue model mice. Therefore, the multi-factor induction method used in this experiment successfully constructed a fatigue mouse model. The PAP intervention group and the Rhodiola rosea positive control group showed similar effects in improving fatigue-related indicators, suggesting that PAP may have anti-fatigue efficacy comparable to Rhodiola rosea.

[0151] (5) Liver glycogen (LG) and muscle glycogen (MG) in mice after exercise: During exercise, when blood glucose levels decrease, the body breaks down liver glycogen into glucose. Simultaneously, some glucose enters muscle cells to synthesize muscle glycogen, directly supplying energy to muscle tissue. Therefore, glycogen content is an important indicator for assessing fatigue capacity. Figure 12 As shown, after 28 days of modeling treatment, compared with the blank control group, the liver glycogen and muscle glycogen contents of mice in the model group were significantly reduced (p<0.001), reaching 2.49±0.57 mg / mL and 2.18±0.11 mg / mL, respectively. This result confirms the successful establishment of the fatigue mouse model. The experimental results after drug intervention showed that the liver glycogen (low dose: 2.90±0.47 mg / mL; high dose: 3.95±0.39 mg / mL; positive group: 3.99±0.5 mg / mL) and muscle glycogen (low dose: 3.22±0.51 mg / mL; high dose: 3.76±0.53 mg / mL; positive group: 4.14±0.27 mg / mL) levels in the Rhodiola rosea and PAP treatment groups were significantly increased compared with the model group (p<0.05), indicating that both drugs can effectively improve the glycogen reserve function of fatigued mice. Therefore, the multifactorial induction method used in this experiment successfully constructed a fatigue mouse model. The PAP intervention group and the Rhodiola rosea positive control group showed similar effects in improving fatigue-related indicators, indicating that PAP may have anti-fatigue efficacy comparable to Rhodiola rosea.

[0152] (6) Malondialdehyde (MDA), superoxide dismutase (SOD), and glutathione peroxidase (GSH-px) in mouse muscles after exercise: (e.g.) Figure 13 As shown, after 28 days of modeling and drug administration, the detection of oxidative stress indicators showed that the serum MDA level in the model group mice was significantly higher than that in the blank group (p<0.0001), reaching 12.16±0.52 nmol / L. This result further verified the reliability of the establishment of the fatigue mouse model. Drug intervention results showed that, compared with the model group, the MDA levels in the Rhodiola rosea group and each PAP dose group (low dose: 11.26±0.49 nmol / L; high dose: 10.34±0.32 nmol / L; positive group: 9.83±0.32 nmol / L) were significantly lower (p<0.05). In the body, the presence of reactive oxygen species leads to protein oxidation. Superoxide dismutase (SOD) can catalyze the generation of hydrogen peroxide and oxygen from reactive oxygen species in the body, and glutathione peroxidase (GSH-px) can further catalyze the generation of water and oxygen from hydrogen peroxide, protecting body tissues from free radical attack. Therefore, increasing the enzyme activity of SOD and GSH-px can help relieve fatigue. Figure 13As shown, after 28 days of multifactorial induction modeling, the levels of SOD and GSH-px in the fatigue model group mice decreased significantly (p<0.0001), reaching 38.48±2.24 pg / mL and 91.71±2.24 pg / mL, respectively. In contrast, the levels of SOD (low dose: 43.91±1.91 pg / mL; high dose: 57.38±2.24 pg / mL; positive group: 66.75±2.24 pg / mL) and GSH-px (low dose: 107.76±9.76 mg / L; high dose: 112.33±4.2 mg / L; positive group: 130.5±4.27 mg / L) in the dose groups increased significantly (p<0.05). It is speculated that PAP can alleviate fatigue by increasing the activity of antioxidant enzymes in the body.

[0153] (7) Muscle structure after exercise in mice: The gastrocnemius muscle of the hind leg of mice was dissected, and histological changes in the gastrocnemius muscle were observed by HE staining to reflect the mouse's exercise capacity. Figure 14 As shown, compared with the control group, many skeletal muscle cells in the model group mice were loosely arranged, with ruptured nuclei, numerous intracellular cracks, and severe necrosis. In the Rhodiola rosea-positive group, local muscle fibers showed cracks. In the low-dose administration group, skeletal muscle cells were loosely arranged, with many cells showing slight swelling and some cracks. In contrast, the gastrocnemius muscle cells in the high-dose administration group were tightly arranged, with only slight cracks. Therefore, Rhodiola rosea and PAP can protect the gastrocnemius muscle of mice to some extent, relieve fatigue, and enhance motor function.

[0154] In summary, PAP can alleviate peripheral fatigue by increasing muscle mass, forelimb grip strength, improving exercise endurance, reducing BUN content, reducing LDH activity, increasing LG and MG content, reducing MDA content, enhancing SOD and GSH-px activity, and reducing gastrocnemius muscle damage in fatigued mice.

[0155] 6.4.2 Relieving Central Nervous System Fatigue

[0156] (1) Neurotransmitters in the mouse hypothalamus: Under resting conditions, serotonin (5-HT), dopamine (DA), and norepinephrine (NE) in the mouse brain are basically in dynamic equilibrium. The stability of their levels can indirectly reflect the degree of neuronal excitability, while their levels decrease under fatigue conditions. Figure 15As shown, after 28 days of modeling and drug administration, the neurotransmitter detection results showed that, compared with the blank control group, the levels of serotonin (170.28±12.86pg / mL), dopamine (31.78±2.74pg / mL), and norepinephrine (94.92±7.43pg / mL) in the brain of the model group mice were significantly reduced (p<0.0001). This result confirms from a neurological perspective that the multifactor-induced fatigue mouse model was successfully constructed. Drug intervention experiments showed that the levels of 5-HT (low dose: 214.06±13.06 pg / mL; high dose: 225.32±6.39 pg / mL; positive group: 266.05±3.58 pg / mL) in mice treated with Rhodiola rosea and PAP were significantly higher than those in the model group (p<0.0001), and the levels of DA (36.73±1.06 pg / mL in the high dose group and 41.06±2.48 pg / mL in the positive group) were significantly higher than those in the model group. The levels of 6.62±3.61 pg / mL in the low-dose group were significantly higher than those in the model group (p<0.001), and the levels of NE (low-dose: 139.89±6.02 pg / mL; high-dose: 151.69±6.48 pg / mL; positive group: 153.36±5.76 pg / mL) were also significantly higher than those in the model group (p<0.0001), suggesting that both drugs can effectively regulate neurotransmitter metabolism disorders in fatigued mice.

[0157] (2) Open Field Experiment in Mice: In the open field experiment, a decrease in the number of times an animal traverses a grid usually indicates a decline in its activity level, possibly due to central nervous system fatigue leading to weakened motor coordination and motivation. An increase in the number of cheek scratches can reflect anxiety or discomfort behaviors caused by fatigue; central nervous system fatigue affects mood regulation, leading to an increase in such behaviors. Central nervous system fatigue affects curiosity and motor ability, resulting in a decrease in upright behavior. These behavioral changes are closely related to central nervous system fatigue; fatigue affects motor, mood, and exploratory abilities, thereby altering the performance of these behaviors. Therefore, by observing these indicators, the degree of fatigue in the central nervous system can be indirectly assessed. The results of the open field experiment are derived from... Figure 16Before drug administration, there were no statistically significant differences among the groups of mice in terms of autonomous behavior indicators (number of squares crossed, number of times standing upright on both legs, and number of times they scratched their cheeks for modification) (p>0.05). After 28 days of modeling, the number of squares crossed by the model group (126±29) was significantly reduced compared to the control group (221±45) (p<0.001), indicating that the multi-factor induction method successfully constructed a fatigue animal model with reduced autonomous behavior. After drug intervention, the number of squares crossed by mice in the Rhodiola rosea positive group (181±54) and the high-dose PAP group (182±61) was significantly increased compared to the model group (p<0.05). On day 28 after modeling, the results showed that the number of times mice in the model group stood upright on both legs (13±5 times) was significantly reduced compared to the control group (28±12 times) (p<0.01), further validating the successful establishment of the fatigue model. In the drug intervention groups, the number of times the Rhodiola rosea-positive group (24±18 times) and the high-dose PAP group (23±12 times) were significantly increased compared with the model group (p<0.05), indicating that PAP intervention can effectively improve fatigue-induced behavioral inhibition. There was no statistically significant difference in the number of cheek-grabbing modifications among the groups (p>0.05), indicating that PAP has a limited effect on the modification behavior of mice.

[0158] (3) Mouse tail suspension test: Central nervous system fatigue leads to a decrease in the animal's responsiveness to external stimuli, manifested as a significant increase in immobility time during the tail suspension test. Figure 17 Before drug administration, on day 0, the average immobility time of mice in the five groups was 27.55±7.86 s, with no significant difference (p>0.05). After 28 days of drug administration, behavioral analysis showed that compared with the blank control group, the immobility time in the model group (72±19 s) was significantly increased (p<0.0001), validating the model's effectiveness. Drug intervention results showed that the immobility time of mice in the Rhodiola rosea positive group (40±11 s), low-dose group (50±17 s), and high-dose PAP group (41±14 s) was significantly shorter than that in the model group (p<0.01), indicating that PAP has a significant ameliorative effect on fatigue-induced psychobehavioral inhibition.

[0159] (4) Mouse hippocampus tissue: Histopathological results are as follows Figure 18As shown, the neurons in the hippocampus of the control group were densely packed, neatly arranged, and had clearly visible nuclei, with no obvious pathological changes. The model group exhibited typical characteristics of neuronal injury, with disordered neuronal arrangement, widened intercellular spaces, cell swelling accompanied by deep cytoplasmic staining, and a significantly thinned granule cell layer with abundant inflammatory cell infiltration. These pathological changes indicate that the fatigue model successfully induced hippocampal tissue damage. The Rhodiola rosea group and the PAP group showed some improvement in neuronal state compared to the model group, with increased neuronal density, reduced cytoplasmic staining, and less granule cell loss. Therefore, the multifactorial induction method can establish a stable fatigue-related hippocampal injury model, and PAP intervention can improve neuronal morphology with similar effects to Rhodiola rosea.

[0160] In summary, PAP can increase the number of grids traversed in the open field test, increase the number of times bipedalism is achieved, reduce the time of tail suspension immobility, upregulate 5-HT, DA and NE levels, and improve hippocampal neuronal damage, thereby alleviating central nervous system fatigue.

[0161] 6.4.3 Correlation analysis of antioxidant and anti-fatigue effects

[0162] There are various theories about the mechanisms of fatigue, and the "free radical scavenging theory" is currently the only one that can be investigated both in vivo and in vitro. Pearson correlation analysis of in vivo antioxidant and anti-fatigue indicators revealed a correlation between anti-fatigue and antioxidant activity in mice after PAP dosage intervention. Chlamydomonas reinhardtii protein peptides may exert their anti-fatigue effect by increasing the activity of antioxidant enzymes and protecting the body from oxidative stress damage. Figure 19 As shown, although the antioxidant and anti-fatigue effects did not show significant changes in this study, possibly due to the small sample size, the analysis revealed that the absolute value of the Pearson correlation coefficient between antioxidant and anti-fatigue effects was greater than 0.7. The larger the absolute value of the correlation coefficient, the stronger the correlation. Therefore, antioxidant and anti-fatigue effects are still correlated.

[0163] The results showed that MDA was positively correlated with LDH and negatively correlated with MG and BUN, with correlation coefficients of -0.717, 0.986, and -0.974 for MDA content and MG, LDH, and BUN, respectively. SOD was positively correlated with exhaustive swimming time, 5-HT, and NE, with correlation coefficients of 0.715, 0.939, and 0.896 for SOD content and exhaustive swimming time, respectively. GSH-px content was negatively correlated with exhaustive swimming time, with a correlation coefficient of -0.759. Therefore, these results indicate that the anti-fatigue activity and antioxidant activity of PAP in mice are correlated, and also demonstrate the feasibility of using antioxidant indicators as evaluation indicators for screening Chlamydomonas reinhardtii protein peptides.

[0164] In summary, the advantage of this invention lies in its intelligent formulation design for a ternary solid beverage combining Chlamydomonas reinhardtii, matcha, and coffee by combining the Grey Wolf optimization algorithm with a backpropagation neural network. This not only enhances the product's functionality and stability but also expands the application avenues for microalgae proteins in high-value-added foods. This method can simultaneously optimize multiple key indicators, including sensory evaluation, physicochemical properties, antioxidant activity, and anti-fatigue effects, thereby significantly enhancing the product's functionality while ensuring its taste and flavor. This provides a new technological path for the intelligent manufacturing of functional foods.

[0165] The beneficial effects of this invention lie in the significant advantages that the newly developed anti-fatigue coffee compound solid beverage exhibits in anti-fatigue, antioxidant, and neuroprotective properties. It effectively regulates peripheral energy metabolism, improves central nervous system function, and eliminates excess free radicals, thereby alleviating the various negative effects of exercise fatigue. Furthermore, the product is convenient to carry, quick to prepare, and has flexible formulations, facilitating market promotion and consumer acceptance, and contributing to the popularization and upgrading of functional foods.

Claims

1. A novel anti-fatigue coffee solid beverage, characterized in that, The coffee solid beverage comprises the following components: coffee extract concentrate, Chlorella vulgaris protein peptide PAP, matcha, fresh milk creamer and sucrose.

2. A process for the preparation of the novel anti-fatigue coffee solid beverage as claimed in claim 1, characterized in that, The method comprises the following steps: (1) A certain amount of coffee powder is weighed and extracted with water in a certain proportion to prepare a coffee extract concentrate; (2) The coffee extract concentrate, Chlorella vulgaris protein peptide PAP, matcha powder, fresh milk creamer and sucrose syrup are added to the concentrate in a certain proportion for blending; (3) The blended concentrate is homogenized by a high-speed dispersion homogenizer; (4) The blended liquid is pre-frozen and freeze-dried to obtain a coffee solid beverage freeze-dried powder; (5) The freeze-dried powder is sterilized by a pasteurizer; (6) The ABTS clearance rate of the coffee solid beverage is determined and the coffee solid beverage is subjected to sensory evaluation; (7) Vacuum packaging and low-temperature preservation; (8) The obtained data are input into a BP neural network, and a grey wolf algorithm is used to find the best production formula, and the formula is verified.

3. The process for the preparation of the new anti-fatigue coffee solid beverage according to claim 2, characterized by the fact that, In the step (1), the coffee powder and water are in a proportion of 1:2 (g / mL) for extraction.

4. The process for the preparation of the new anti-fatigue coffee solid beverage according to claim 2, characterized by the fact that, In the step (2), the components are prepared in a certain proportion by mass fraction, including 8-16 parts of coffee extract concentrate, 3-7 parts of Chlorella vulgaris protein peptide PAP, 1-5 parts of matcha powder, 50-90 parts of fresh milk creamer and 1-5 parts of sucrose.

5. The method of preparing the novel anti-fatigue coffee solid beverage according to claim 2, characterized in that, In the step (2), the Chlorella vulgaris protein peptide PAP is prepared as follows: (1) Extraction of Chlorella vulgaris protein ① Chlorella vulgaris powder is mixed with DES reagent with a water content of 72.23% by mass fraction at a solid-liquid ratio of 1:10 (g / mL); ② Alkaline dissolution: the pH is adjusted to about 9.5 with NaOH, and after low-temperature swelling and ultrasonic treatment, the supernatant is collected by centrifugation; ③ Acid precipitation: 1-fold 60% ammonium sulfate solution and 2-fold saturated sodium sulfate solution are added to the supernatant, and after mixing, the pH is adjusted to about 3.5, and the precipitate is collected by centrifugation after low-temperature standing. The precipitate is redissolved in ultrapure water, dialyzed and freeze-dried to obtain Chlorella vulgaris protein; (2) The Chlorella vulgaris protein is prepared into a protein solution with ultrapure water, and after mixing in a water bath, the pH is adjusted to about 6.

0. An appropriate amount of papain is added, and the pH is kept stable at about 55°C for enzymolysis. After inactivation in a boiling water bath, freeze-drying is performed to obtain Chlorella vulgaris protein peptide PAP.

6. The process for the preparation of the new anti-fatigue coffee solid beverage according to claim 5, characterized by the fact that, The DES reagent is composed of choline chloride and glycerol in a volume ratio of 1:

1.

7. The method of preparing the novel anti-fatigue coffee solid beverage according to claim 5, characterized in that, The Chlorella vulgaris protein peptide PAP is purified to obtain three ultrafiltration components: PAP-I with a molecular weight of >10k, PAP-II with a molecular weight of <10k and PAP-III with a molecular weight of <3k. The component with the strongest ABTS free radical scavenging capacity is selected for polypeptide sequence identification and screening.

8. The preparation method of the novel anti-fatigue coffee solid beverage according to claim 5, characterized in that, The Chlorella vulgaris protein peptide PAP has a significant effect on anti-fatigue in mice, mainly in relieving peripheral fatigue and central nervous fatigue.

9. The preparation method of the novel anti-fatigue coffee solid beverage according to claim 5, characterized in that, The Chlorella vulgaris protein peptide PAP realizes the anti-fatigue effect by regulating peripheral energy metabolism, improving central nervous system function and scavenging excess free radicals.

10. The method of preparing the novel anti-fatigue coffee solid beverage according to claim 2, characterized in that, In the step (6), during sensory evaluation, each package of freeze-dried powder is mixed with water in a mass-volume ratio of 1:20 (g / mL).

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