Preparation method of oyster peptide and application thereof

Abstract

The application discloses a preparation method of oyster peptide and application thereof and belongs to the technical field of marine bioactive substances. The preparation method of the oyster peptide in the oyster peptide energy drink is as follows: fresh edible parts of oysters are homogenized after being frozen, 0.05wt%-0.3wt% animal compound protease and 0.01wt%-0.2wt% neutral protease are added, and enzymolysis is carried out for 1-7 hours to obtain oyster peptide crude extract; the oyster peptide crude extract is subjected to decolorization treatment, then filtration, the filtrate after filtration is subjected to desalting treatment, and the solution after desalting is subjected to spray drying treatment to obtain oyster peptide powder. The application optimizes the preparation process of the oyster peptide, obtains peptide powder with better flavor and stability in beverage application, and solves the problem of reverse odor and precipitation of the oyster peptide in beverage products on the market.

Description

Technical Field

[0001] This invention belongs to the field of marine bioactive substances technology, and more specifically, relates to a method for preparing oyster peptides and their applications. Background Technology

[0002] In general, most people selectively ignore the sub-healthy state without any organic lesions, thus creating hidden dangers for it to eventually develop into a disease state. Therefore, regulating bodily functions, getting rid of the sub-healthy state, and controlling the occurrence and development of diseases from the source should be given our attention.

[0003] Currently, most energy drinks on the market contain taurine, caffeine, guarana, white sugar, vitamins, and minerals. These drinks are effective for energizing working adults and those prone to fatigue from high-intensity work. However, for students whose bodies are not yet fully developed, their ability to metabolize caffeine is weak, and even small amounts can reach toxic doses and cause harm. Furthermore, the taurine in these drinks is mostly synthetic and lacks the biological activity of natural taurine. Studies show that taurine is primarily metabolized by the kidneys after absorption to maintain a balance in the body; excessive intake will inevitably burden the kidneys. Frequent and excessive consumption of these drinks can easily lead to dependence and significant side effects. Additionally, the large amounts of added white sugar are unsuitable for people trying to lose weight, those with impaired blood sugar regulation, and those with type 2 diabetes. Therefore, the market urgently needs a new type of energy drink that fundamentally addresses these pain points, caters to market trends, meets consumer needs, uses natural and safe ingredients, and offers functions such as anti-fatigue, antioxidant, energy replenishment, and energizing / calming effects.

[0004] However, energy peptide beverages on the market often exhibit severe off-flavor and turbidity, with poor product flavor, especially after 8 months of shelf life. They typically show a large amount of visible flocculation and sediment, as well as unbearable characteristic flavor and fishy smell. Summary of the Invention

[0005] The purpose of this invention is to provide a method for preparing oyster peptides and their applications, in order to overcome the shortcomings of the prior art.

[0006] To achieve the above objectives, the present invention is implemented through the following technical solution:

[0007] A method for preparing oyster peptides, wherein the edible parts of oysters are enzymatically hydrolyzed, and the enzymes selected are animal complex protease and neutral protease.

[0008] Furthermore, the preparation method specifically involves: freezing and homogenizing the edible parts of fresh oysters, adding 0.05%–0.3 wt% animal complex protease and 0.01%–0.2 wt% neutral protease, and enzymatically hydrolyzing for 1–7 hours to obtain a crude oyster peptide extract; decolorizing the crude peptide extract, then filtering it, desalting the filtrate, and spray-drying the desalted solution to obtain oyster peptides.

[0009] Furthermore, the animal complex protease includes trypsin, pepsin, and alkaline protease; the mass fractions of the three enzymes added are: trypsin 0.025%–0.15 wt%, pepsin 0.015%–0.1 wt%, and alkaline protease 0.01%–0.05 wt%.

[0010] Furthermore, the desalination process employs membrane separation technology.

[0011] The application of the oyster peptide in the preparation of beverage products.

[0012] The application of the oyster peptide in the preparation of energy drinks with anti-fatigue function.

[0013] An energy drink with anti-fatigue properties comprises the following ingredients by mass fraction: 75-90 parts drinking water, 5-8 parts oyster peptides, 1-3 parts natural taurine, 2-5 parts inositol, 3-8 parts niacinamide, 0.02-0.05 parts vitamin B6, and vitamin B... 12 The oyster peptide contains 0.03-0.06 parts of dietary fiber, 1-3 parts of sweetener, 5-20 parts of sweetener, and 0.1-0.3 parts of acidity regulator; the preparation method of the oyster peptide is as described above.

[0014] The advantages and beneficial effects of this invention are as follows:

[0015] This invention, through screening proteases and determining enzyme cleavage sites and hydrolysis endpoints, prepares protein hydrolysates with higher exposure of bitter amino acids and lower exposure of umami amino acids. Combined with membrane separation technology, this yields oyster peptide powder with high purity, good flavor, low fat content, and no subsequent fishy odor, thus facilitating the wider application of oyster peptides as a food ingredient. This invention optimizes the preparation process of oyster peptide powder, resulting in a peptide powder with better flavor and stability in beverage applications, and solving the problem of fishy odor and sedimentation currently found in commercially available oyster peptide powders used in beverage products.

[0016] This invention utilizes raw materials rich in natural taurine, employing a scientific formula and a relatively simple production process to address the problems of existing energy drinks, such as high sugar content, caffeine dependence, and a lack of consideration for human health. This invention uses naturally extracted oyster peptides to effectively alleviate fatigue, improve nutrient digestion and absorption, and enhance energy utilization, thereby alleviating sub-health conditions such as lethargy. The energy drink formula with anti-fatigue function provided by this invention has been certified as low-GI by a third-party testing agency. Preliminary experimental evaluation shows its GI value to be between 30 and 40 (≤55), making it widely applicable to various groups, such as students, the elderly, those trying to lose weight, and people with type 2 diabetes. Detailed Implementation

[0017] The technical solution of the present invention will be further described and illustrated below with reference to specific embodiments.

[0018] When developing oyster peptide beverages, in addition to ensuring their functionality and digestibility, it is also necessary to improve the preparation process to avoid problems such as poor flavor and unstable system during the shelf life of the beverage.

[0019] The basic flavor profile of peptides manifests in five categories: sour, sweet, bitter, salty, and umami. For the same raw material, the specificity of different enzymes and their cleavage sites significantly affect the molecular weight and amino acid composition of the enzymatic hydrolysates, leading to strong sensory differences. Oyster peptides have a delicious meaty flavor and are rich in flavor-enhancing amino acids (glutamic acid, aspartic acid, glycine, and alanine). Studies have found that the proportion of sweet amino acids in their enzymatic hydrolysate is as high as 48.1%, and the proportion of bitter amino acids is as high as 51.9%. Commercially available oyster peptide powders are mostly exposures of small umami-enhancing peptides, but in actual formulation, the richness of umami flavor is inversely proportional to the overall beverage flavor. Furthermore, in traditional beverage sterilization processes, high temperature and pressure or pH < 4 can cause denaturation of proteins and free fatty acids in raw materials, making it difficult to maintain good flavor and stability over shelf life.

[0020] Example 1:

[0021] This embodiment studies the selected enzyme preparation and enzymatic hydrolysis conditions through experimental analysis.

[0022] 1. Enzyme Screening: Five enzymes—neutral protease, alkaline protease, aquatic protease, flavor protease, and animal complex enzyme—were screened and combined in different ways. The enzymatic hydrolysis endpoint was determined by the residue content. After enzyme inactivation treatment, crude oyster peptide extract was obtained for sensory evaluation. Referring to the national standard GB / T 16291.1-2012, the sensory evaluation team consisted of 18 males and 18 females without taste impairment (10 women and 8 men, aged 25-32). The temperature of the evaluation room was controlled at room temperature (25±2℃). For the experimental characteristics, reference solutions were used to train the team members. The reference solutions were as follows: umami (monosodium glutamate solution, 15g / L); sweetness (sucrose solution, 16g / L); sourness (citric acid solution, 1g / L); astringency (quercetin solution, 0.5g / L); bitterness (caffeine, 0.5g / L).

[0023] The characteristic flavor profile is complex; therefore, this evaluation used oyster broth (10g / L, boiled for 20 minutes) as a reference solution. Six reference solutions were used as the standard flavor score, with a maximum of 10 points, to evaluate the relative flavor of the enzymatic hydrolysate. The score ranged from 0 to 10, with higher scores indicating a stronger flavor. The evaluation consisted of two steps: smelling and tasting. The oyster extracts were kept at a constant temperature of 35°C and stored in 20mL disposable plastic cups. Participants first smelled the flavor, then tasted it on the front, middle, back, and sides of their tongue for approximately 15 seconds, spitting it out or swallowing it. They then rinsed their mouths with distilled water and repeated the process after 5 minutes. The final result was the average score from all group members.

[0024] Table 1 Sensory evaluation of different oyster hydrolysates

[0025] variety Umami sour bitterness astringent sweet Characteristic Flavor neutral protease 5 4 8 6 3 4 alkaline protease 7 6 7 4 4 7 Aquatic proteases 9 7 4 8 4 8 Flavor proteases 7 7 3 5 6 9 Animal complex protease 5 5 6 5 3 4

[0026] Note: All ratings have had their highest and lowest scores removed, and the average of the remaining scores is used.

[0027] According to Table 1, the order of freshness from highest to lowest is: aquatic protease > flavor protease = alkaline protease > animal complex protease (trypsin: pepsin: alkaline protease = 5.5: 3.5: 1) = neutral protease.

[0028] The order of bitterness from highest to lowest is: neutral protease > alkaline protease > animal complex protease > aquatic protease > flavor protease

[0029] The characteristic flavor profiles, from highest to lowest, are: flavor protease > aquatic protease > alkaline protease > neutral protease = animal complex protease.

[0030] The aim of this preparation was to obtain oyster hydrolysate with a predominantly bitter taste and a mild umami and characteristic flavor. Therefore, neutral protease and animal complex protease were selected from Table 1.

[0031] Example 2:

[0032] Oysters are manually shelled, and the edible parts are frozen and homogenized. Then, 0.05–0.3% of animal complex protease (containing 0.025%–0.15 wt% trypsin, 0.015%–0.1 wt% pepsin, and 0.01%–0.05 wt% alkaline protease) is added, and the mixture is enzymatically hydrolyzed for 1–7 hours. The enzyme is then inactivated at 95°C to obtain a crude oyster peptide extract. Activated carbon and diatomaceous earth are added to the crude peptide extract, and the mixture is stirred at 50–80°C for 15–20 minutes. The mixture is then filtered through a plate and frame filter. The filtrate is desalted using membrane separation technology, and the desalted solution is spray-dried to obtain the oyster peptide raw material.

[0033] Example 3:

[0034] Oysters are manually shelled, and the edible parts are frozen to form frozen plates. After homogenization, 0.01-0.2% neutral protease is added, and the mixture is enzymatically hydrolyzed for 1-7 hours. The enzyme is then inactivated at 95°C to obtain a crude oyster peptide extract. Activated carbon and diatomaceous earth are added to the crude peptide extract, and the mixture is stirred at 50-80°C for 15-20 minutes. The mixture is then filtered through a plate and frame filter. The filtrate is desalted using membrane separation technology, and the desalted solution is spray-dried to obtain the oyster peptide raw material.

[0035] Example 4:

[0036] Oysters are manually shelled, and the edible parts are frozen and homogenized. Then, 0.05–0.3% animal complex protease (0.025%–0.15 wt% trypsin, 0.015%–0.1 wt% pepsin, 0.01%–0.05 wt% alkaline protease) and 0.01–0.2% neutral protease are added. Enzymatic hydrolysis is performed for 1–7 hours, followed by enzyme inactivation at 95°C to obtain a crude oyster peptide extract. Activated carbon and diatomaceous earth are added to the crude peptide extract, and the mixture is stirred at 50–80°C for 15–20 minutes. The mixture is then filtered through a plate and frame filter. The filtrate is desalted using membrane separation technology, and the desalted solution is spray-dried to obtain the oyster peptide raw material.

[0037] The degree of protein hydrolysis was tested using protein hydrolysis as the evaluation criterion. Related experimental data showed that the degree of protein hydrolysis was directly proportional to the hydrolysis time; however, the stability of the hydrolysate decreased after a certain hydrolysis time. Therefore, evaluating the impact of protein hydrolysis on the product is crucial. The nitrogen content of the oyster extract and hydrolysate was determined using the Kjeldahl method, and the ammonia nitrogen content was determined using formaldehyde titration. The formulas for calculating protein recovery rate and degree of protein hydrolysis are as follows:

[0038] Degree of protein hydrolysis (DH) = (F t -F0) / N×100%

[0039] F tContent of free amino nitrogen in oyster enzymatic hydrolysate (g / 100mL);

[0040] F0: Free amino nitrogen content in oyster extract (before enzymatic hydrolysis) (g / 100mL);

[0041] N: Protein nitrogen content in oyster extract (before enzymatic hydrolysis) (g / 100mL).

[0042] Table 2. Effects of different enzyme preparations and hydrolysis times on the degree of protein hydrolysis.

[0043]

[0044] As shown in Table 2, the degree of protein hydrolysis of the three samples is in the order of Example 4 > Example 2 > Example 3.

[0045] Furthermore, the oyster peptides prepared using the method described in Example 4 exhibit a solubility as high as 99%. Analysis shows that the essential amino acid composition of the oyster peptides reaches 55.7 g / 100 g, higher than that of milk protein (50.0 g / 100 g) and egg protein (49.7 g / 100 g). The polysaccharide content is ≥15.0%, and the natural taurine content is ≥1500 mg / 100 g. It demonstrates good stability, coexistence stability with other components, and good storage stability; low viscosity; good processability; low osmotic pressure and low allergenicity; and good palatability. Therefore, the oyster peptides prepared in Example 4 are suitable for the development of oyster peptide energy drinks.

[0046] In addition, the oyster peptides added in Example 4 are preferably derived from triploid oysters from exclusive natural sea areas, using oysters harvested from October to April of the following year. These oysters are pollution-free, large, meaty, and nutritious. Of course, high-quality oyster peptide powder can also be obtained by using oysters from other production areas and months.

[0047] Example 5:

[0048] An oyster peptide energy drink is made from the following ingredients: 90 parts drinking water, 6 parts oyster peptide powder (commercially available product), 3 parts taurine, 2 parts inositol, 4 parts niacinamide, 0.04 parts vitamin B6, and B... 12 The ingredients are: 0.05 parts dietary fiber, 3 parts sweetener, 20 parts acidity regulator, 0.2 parts edible flavoring, 0.03 parts preservative, 0.04 parts tartrazine, 0.08 parts allura red, and 0.02 parts allura red.

[0049] The preparation method of this oyster peptide energy drink includes the following steps:

[0050] (1) Add the following ingredients to the formula: water (6 parts), oyster peptides (3 parts), taurine (3 parts), inositol (2 parts), nicotinamide (4 parts), vitamin B6 (0.04g), and vitamin B1 (4 parts). 12The following ingredients are added to a mixing bowl in sequence: 0.05 parts dietary fiber, 3 parts sweetener, 20 parts sweetener, 0.2 parts acidity regulator, 0.8 parts pectin, 0.3 parts soybean polysaccharide, 0.03 parts edible flavoring, 0.04 parts preservative, 0.03 parts tartrazine, and 0.07 parts brilliant blue. After mixing for 3-5 minutes, add 0.2 parts acidity regulator at room temperature and mix well.

[0051] (2) After filtration through a filter with a filtration level of 10-50μm, the filtered liquid is transparent, odorless, and free of visible matter.

[0052] (3) Fill the clarified liquid into bottles, sterilize the filled products, and then seal them.

[0053] Example 6:

[0054] An oyster peptide energy drink is made from the following ingredients: 90 parts drinking water, 6 parts oyster peptide powder (prepared according to Example 4), 3 parts taurine, 2 parts inositol, 4 parts nicotinamide, 0.04% B6, and B... 12 The ingredients are: 0.05 parts dietary fiber, 3 parts sweetener, 20 parts acidity regulator, 0.2 parts edible flavoring, 0.03 parts preservative, 0.04 parts tartrazine, 0.08 parts allura red, and 0.02 parts allura red.

[0055] The preparation method of this oyster peptide energy drink includes the following steps:

[0056] (1) Add the following ingredients to the formula: water (6 parts), oyster peptides (3 parts), taurine (3 parts), inositol (2 parts), nicotinamide (4 parts), vitamin B6 (0.04g), and vitamin B1 (4 parts). 12 The following ingredients are added to a mixing bowl in sequence: 0.05 parts dietary fiber, 3 parts sweetener, 20 parts sweetener, 0.2 parts acidity regulator, 0.8 parts pectin, 0.3 parts soybean polysaccharide, 0.03 parts edible flavoring, 0.04 parts preservative, 0.03 parts tartrazine, and 0.07 parts brilliant blue. After mixing for 3-5 minutes, add 0.2 parts acidity regulator at room temperature and mix well.

[0057] (2) After filtration through a filter with a filtration level of 10-50μm, the filtered liquid is transparent, odorless, and free of visible matter.

[0058] (3) Fill the clarified liquid into bottles, sterilize the filled products, and then seal them.

[0059] Evaluation experiments were conducted on Examples 5 and 6:

[0060] 1) Evaluation of the sensory properties of oyster peptide beverages using the paired comparison method

[0061] Referring to the national standard GB / T 16291.1-2012, the sensory evaluation panel of this study selected 16 males and 16 females without taste disorders, including 10 women and 6 men (aged between 25 and 32). The temperature of the evaluation room was controlled at room temperature (25±2℃). The beverages prepared in Examples 5 and 6 were placed at 4℃, 25℃, and 37℃ for 3 months, respectively. The appearance and quantity of the samples were consistent, and one set of samples was given to each taster. The differences in aroma, sweetness, sourness, fishy smell (whether it was fishy), and freshness of the samples were evaluated, and the results were recorded in the evaluation table in Table 3.

[0062] Table 3. Targeted Pairing Comparison Evaluation Table

[0063]

[0064] The following table shows the differences in opinions expressed by 16 tasters regarding two oyster peptide drinks:

[0065] Table 4. Statistical Table of Difference Pairwise Evaluation Results

[0066]

[0067]

[0068] As shown in the table above, only 1 out of 16 respondents indicated no significant difference, while the rest reported a severe fishy smell in Example 1. Regarding aroma, 13 respondents indicated that Example 6 had a better aroma, while 1 person indicated no difference. In terms of sweet and sour taste, since Examples 5 and 6 used the same sweet and sour flavorings, 7 respondents indicated no significant difference, while 7 people indicated that Example 6 had a better sweet and sour taste. Regarding freshness, 14 respondents indicated that Example 6 had a better freshness, while 1 person indicated no significant difference. Therefore, it is evident that the homemade oyster peptide powder has a better flavor over its shelf life.

[0069] To determine the effect of oyster peptide powder on the stability of beverages, the beverages from Examples 5 and 6 were placed at 4°C, 25°C, and 37°C for 3 months, respectively, and their product stability was observed.

[0070] Table 5. Effects of different enzyme preparations and hydrolysis time on the system.

[0071]

[0072]

[0073] In Example 5, at 4°C, the beverage prepared from externally sourced oysters began to precipitate on the third day of the first week; at 25°C, precipitation began to form from the fourth week; and at 37°C, precipitation began to form from the third week. In contrast, Example 6 showed no precipitation at elevated temperatures or over extended periods, indicating that the oyster peptides added in Example 6 made the overall system more stable.

[0074] The oyster peptide powder prepared by the enzymatic hydrolysis method provided in Example 6 has a more delicate and harmonious taste and a more stable and superior flavor in beverage formulation applications. Compared with commercially available oyster peptide products, beverages formulated using this method have a better taste, a more stable system, do not develop a fishy smell during the shelf life, and provide a better consumer experience.

[0075] The above demonstrates that the peptide powder prepared in Example 4 not only has a stable flavor without any off-fishy smell, but also does not precipitate under high temperature and high pressure (sterilization) or pH < 4 (acidity regulator) conditions.

[0076] II. Animal experiments verifying the relief of physical fatigue:

[0077] 1. Materials and Methods

[0078] 1.1 Sample: The oyster peptide energy drink (anhydrous powder formula) prepared in Example 6 was submitted for testing. The submitted sample was a milky white solid. Before use, it was prepared with ultrapure water to the appropriate concentration. The approved net content of the sample was 400g / bag. The recommended daily intake of oyster peptide energy drink is 330ml / 60kg·BW. Storage conditions: 4℃. Solvent: ultrapure water.

[0079] 1.2 Comparative Sample: Red Bull Vitamin Flavored Beverage, manufactured by T.C. Pharmaceutical Industries Co., Ltd. (Thailand). The sample is a yellow liquid, with a recommended daily intake of 250ml per person. Sample batch number: 20221205, shelf life: 12 months. Before use, the vitamin flavored beverage should be rotary evaporated at low temperature (50℃) to 1000ml for later use.

[0080] 1.2 Experimental animals: 168 SPF-grade ICR mice; male; initial weight range: 19-23g

[0081] 1.3 Dosage Selection and Administration of the Test Substance: A negative control group (ultrapure water) was set up. Oyster peptide energy drink had three dosage groups: low-dose (14.0 ml / kg·BW), medium-dose (46.7 ml / kg·BW), and high-dose (140.0 ml / kg·BW) (equivalent to 3, 10, and 30 times the recommended daily intake, respectively). Red Bull vitamin flavored beverage also had three dosage groups: low-dose (12.5 ml / kg·BW), medium-dose (41.7 ml / kg·BW), and high-dose (125 ml / kg·BW) (equivalent to 3, 10, and 30 times the recommended daily intake, respectively). The test substance and control samples were administered orally at a dose of 0.2 ml / 10 g·BW.

[0082] 1.4 Main Instruments and Reagents: Swimming tank (50cm×50cm×40cm), SPN3001F electronic balance, lead sheet, stopwatch, Hitachi 7020 fully automated biochemical analyzer, UV2100 ultraviolet spectrophotometer, centrifuge, constant temperature water bath; lactate analyzer (Lactate SCOUT), lactate test strips (Lactate Scout Sensors Big Pack 72+, batch number: 1909827254), liver glycogen / muscle glycogen assay kit (BB-4723, Shanghai Beibo Biotechnology Co., Ltd.), urea nitrogen assay kit (MAKER biochemical kit).

[0083] 1.5 Experimental Methods: Grouping: 280 animals were divided into 4 subgroups, with 70 animals in each subgroup. Each subgroup was randomly divided into 7 groups according to body weight: a negative control group, low-dose oyster peptide energy drink groups, medium-dose groups, high-dose groups, and low-dose, medium-dose, and high-dose Red Bull vitamin flavored beverage groups, with 10 animals in each group. A certain amount of concentrated oyster peptide energy drink powder was weighed and dissolved in ultrapure water to prepare a solution of the required concentration. Red Bull vitamin flavored beverage was rotary evaporated at low temperature (50℃) to obtain 2000ml, which was used as the gavage solution for the high-dose group, and then diluted with ultrapure water to prepare a solution of the corresponding concentration. Animals in each group were given different doses of oyster peptide energy drink and Red Bull vitamin flavored beverage by gavage, while the negative control group was given ultrapure water. The gavage volume for mice was 0.2ml / 10g·BW. Gavage was performed once daily at the same time. The gavage volume was adjusted weekly by weighing, and the gavage was continued for 30 consecutive days. Mice in all groups were fed normal feed with free access to food and water. Thirty days later, the mice in each subgroup were tested for their ability to alleviate physical fatigue.

[0084] 1.5.1 Weight-bearing swimming experiment: 30 minutes after the last administration of the test sample to each dose group in subgroup 1, lead sheets with 5% of the mouse body weight were loaded at the base of the mouse tail. The mice were then placed in a swimming tank (water depth ≥ 30 cm, water temperature 25℃ ± 1.0℃) to swim. The time from the start of swimming to death was recorded as the weight-bearing swimming time of the mice.

[0085] 1.5.2 Serum urea nitrogen determination: 30 min after the last administration of the test substance to each dose group in subgroup 2, the mice swam in water at 30℃ without load for 90 min, rested for 60 min, and then blood was collected to separate serum. Serum urea nitrogen was determined using a Hitachi 7020 fully automated biochemical analyzer and MAKER biochemical kits.

[0086] 1.5.3 Liver glycogen assay: Ten mice in each dose group of subgroup 3 were sacrificed 30 minutes after the last administration of the test substance. The liver was taken, rinsed with physiological saline, dried with filter paper, and 0.1-0.2g of liver was accurately weighed and tested according to the kit instructions.

[0087] 1.5.4 Lactate Measurement: 30 minutes after the last gavage in each dose group of subgroup 4, a 1-2 mm sample was taken from the tail of each mouse. The first drop of blood was wiped away with a cotton ball, and a small amount of tail blood was collected. The blood lactate content of the mice at rest was measured using a lactate analyzer. Then, the mice swam in water at 30℃ for 10 minutes without any load. The blood lactate levels were measured immediately after exercise and at rest at 0 min and 20 min after swimming, respectively, using a lactate analyzer.

[0088] 1.6 Statistical Analysis of Experimental Data: Analysis of variance was used to compare the differences in animal body weight, blood urea nitrogen, liver glycogen, and blood lactate (before swimming, 0 min after swimming, and 20 min after swimming) between the experimental groups and the negative control group. p < 0.05 was considered statistically significant. Due to unequal variances, the weighted swimming time and area under the blood lactate curve were transformed using reciprocal transformation and COS transformation, respectively, before analysis of variance was used to compare the differences between the experimental groups and the negative control group. p < 0.05 was considered statistically significant.

[0089] 1.7 Result Interpretation: Weight-bearing swimming time: If the weight-bearing swimming time of the test sample group is significantly longer than that of the control group, and the difference is statistically significant, the experimental result can be considered positive. Blood urea nitrogen: If the serum urea level of the test sample group is lower than that of the negative control group, and the difference is statistically significant, the experimental result can be considered positive. Liver glycogen: If the liver glycogen content of the test sample group is significantly higher than that of the negative control group, and the difference is statistically significant, the experimental result can be considered positive. Blood lactate: The area under the blood lactate curve at the three time points is used as the judgment criterion. If the area under the curve of any test group is smaller than that of the negative control group, and the difference is statistically significant, the experimental result can be considered positive. A positive weight-bearing swimming test result, and positive results for any two of the three biochemical indicators (blood lactate, serum urea, and liver glycogen), indicates that the test sample has the function of relieving physical fatigue.

[0090] 2. Results:

[0091] 2.1 Animal weight gain

[0092] Table 6 shows that the high-dose groups in subgroups 2 and 4 (p < 0.05) had a significant effect during the feeding of oyster peptide energy drinks, while the remaining subgroups showed no significant difference compared to the negative control. Therefore, gavage administration of oyster peptide energy drinks and Red Bull vitamin drinks was not the main factor affecting the significant weight gain of mice, and there was no dose-response relationship between the two. In conclusion, oyster peptide energy drinks and Red Bull vitamin drinks had no effect on the weight of mice during the 30-day gavage experiment.

[0093] Table 6. Effects of oyster peptide energy drink on body weight gain in mice (g, )

[0094]

[0095]

[0096] Note: * indicates a significant difference compared to the negative control group (p<0.05);

[0097] ** indicates a significant difference compared to the negative control group (p<0.01).

[0098] A. Oyster Peptide Energy Drink

[0099] B. Red Bull Vitamin Flavored Drink

[0100] Table 7. Effects of oyster peptide energy drink on weight-bearing swimming time in mice.

[0101]

[0102] Note: ** indicates a statistically significant difference compared to the negative control group (p<0.01).

[0103] Animal experiments have shown that after 30 consecutive days of gavage administration, mice in the low-dose oyster peptide energy drink group and the medium-dose Red Bull vitamin flavored beverage group had significantly longer weight-bearing swimming times than the negative control group (p<0.01). Furthermore, the weight-bearing swimming time of the low-dose oyster peptide energy drink group was 8.65±3.75 min, which was higher than that of the medium-dose Red Bull vitamin flavored beverage group (7.66±3.16 min), indicating a significant prolongation of weight-bearing swimming time in mice.

[0104] Table 8. Effects of oyster peptide energy drink on serum urea nitrogen and liver glycogen in mice.

[0105]

[0106]

[0107] Note: ** The . indicates a significant difference compared to the negative control group (p<0.01).

[0108] After 30 days of gavage administration of oyster peptide energy drink and Red Bull vitamin drink, compared with the negative control group, the high-dose group of mice swam without load for 90 minutes and rested for 60 minutes, and the serum urea nitrogen level was significantly lower (p<0.01); the serum urea nitrogen level of the high-dose oyster peptide energy drink group was 7.3±1.3mmol / L and that of the Red Bull vitamin drink group was 7.9±2.2mmol / L (see Table 8).

[0109] Effects of oyster peptide energy drink on liver glycogen: After 30 days of administration of oyster peptide energy drink, there was no significant change in liver glycogen levels in mice of each dose group compared with the negative control group; after 30 days of administration of Red Bull vitamin flavored beverage, the liver glycogen levels in the medium-dose and high-dose groups of mice were significantly reduced compared with the negative control group, and the differences were statistically significant (p<0.01) (see Table 8).

[0110] Table 9. Effects of oyster peptide energy drink on blood lactate levels in mice (mmol / L)

[0111]

[0112] Note: * The . indicates a significant difference compared to the negative control group (p<0.05);

[0113] ** The . indicates a significant difference compared to the negative control group (p<0.01).

[0114] a After logarithmic transformation, the data are normally distributed with homogeneous variances in each group, and one-way ANOVA is used.

[0115] b Even after logarithmic transformation, the data is still non-normally distributed, so rank-sum analysis is used.

[0116] Animal experiments have shown that after mice were administered 14.0, 46.7, and 140.0 ml / kg·BW oyster peptide energy drink by gavage for 30 consecutive days, the mice's weight-bearing swimming time was significantly prolonged; serum urea nitrogen levels were significantly reduced; and the area under the curve of blood lactate was significantly reduced at three time points: before swimming, 0 min after swimming, and 20 min after swimming. However, no significant effect was observed on the liver glycogen levels of mice at rest. Based on the criteria for relieving physical fatigue in the Ministry of Health's "Technical Specifications for Inspection and Evaluation of Health Foods" (2003 edition), it can be considered that marine active peptide energy drink has the function of relieving physical fatigue.

[0117] The above description is merely a preferred embodiment of the present invention and is not intended to limit the invention in any other way. Any person skilled in the art can make changes or modifications to the above technical content to create equivalent embodiments. However, any simple modifications, equivalent changes, and modifications made to the above embodiments based on the technical essence of the present invention without departing from the scope of the present invention shall still fall within the protection scope of the present invention.

Claims

1. A method for preparing oyster peptides, characterized in that, This method involves enzymatically hydrolyzing the edible parts of oysters using animal complex proteases and neutral proteases. The animal complex proteases include trypsin, pepsin, and alkaline proteases. Specifically, the edible parts of fresh oysters are frozen and homogenized, then 0.05%–0.3 wt% animal complex proteases and 0.01%–0.2 wt% neutral proteases are added, and the mixture is hydrolyzed for 1–7 hours to obtain a crude oyster peptide extract. The crude peptide extract is then decolorized, filtered, and the filtrate is desalted. The desalted solution is then spray-dried to obtain oyster peptides. The 0.05%–0.3 wt% animal complex proteases include 0.025%–0.15 wt% trypsin, 0.015%–0.1 wt% pepsin, and 0.01%–0.05 wt% alkaline proteases.

2. The preparation method according to claim 1, characterized in that, The desalination process employs membrane separation technology.

3. The application of the oyster peptide prepared according to claim 1 in beverage products.

4. The application of the oyster peptide prepared according to claim 1 in a functional beverage for relieving physical fatigue.

5. A functional beverage for relieving physical fatigue, characterized in that, The raw materials include the following components by mass fraction: 75-90 parts drinking water, 5-8 parts oyster peptide prepared according to claim 1, 1-3 parts natural taurine, 2-5 parts inositol, 3-8 parts nicotinamide, 0.02-0.05 parts vitamin B6, and vitamin B... 12 It contains 0.03-0.06 servings of dietary fiber and 1-3 servings of dietary fiber.

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