Turtle protein peptide anti-fatigue oral liquid based on enzymatic hydrolysis and freeze-drying process and its preparation method

Abstract

This invention relates to the field of health product technology, specifically to a turtle protein peptide anti-fatigue oral liquid based on enzymatic hydrolysis and freeze-drying technology and its preparation method. The preparation method includes: Step 1: Preparing medicinal and edible enzymatic hydrolysate: Raw materials include: Rehmannia glutinosa, Astragalus membranaceus, Polygonatum sibiricum, Lycium barbarum, Citrus medica, Ginseng, Cistanche deserticola, and Rosa rugosa; the medicinal and edible enzymatic hydrolysate is prepared using an enzymatic hydrolysis and freeze-drying process; Step 2: Preparing small molecule turtle peptide, sweetener, acidulant, vitamins, and water; Step 3: Mixing the medicinal and edible enzymatic hydrolysate, small molecule turtle peptide, sweetener, acidulant, vitamins, and water, filtering, sterilizing, and filling to obtain the oral liquid. This invention is the first to combine turtle protein peptide (small molecule turtle peptide) with medicinal and edible enzymatic hydrolysate obtained by enzymatic hydrolysis and freeze-drying to prepare an anti-fatigue oral liquid. The oral liquid exhibits good low-temperature and high-temperature stability, stable product performance, and excellent anti-fatigue effect.

Description

Technical Field

[0001] This invention relates to the field of health product technology, specifically to a turtle protein peptide anti-fatigue oral liquid based on enzymatic hydrolysis freeze-drying process and its preparation method. Background Technology

[0002] Oral health supplements are health products in liquid form, typically composed of various natural or synthetic nutrients, herbal extracts, vitamins, minerals, etc. Their purpose is to help consumers supplement nutrients that may be lacking in their daily diet, enhance immunity, improve health, or provide support for specific health problems through oral intake.

[0003] Anti-fatigue refers to a series of methods or products used to help the body relieve or eliminate fatigue caused by excessive physical, mental, or psychological stress, restoring physical and mental vitality. Fatigue is a common physiological and psychological phenomenon, typically manifesting as physical weakness, lethargy, poor concentration, and decreased work efficiency. Anti-fatigue oral liquids are currently a popular category of health supplements, providing the body with nutrition, improving concentration, and promoting overall health.

[0004] Turtle protein peptides (small molecule turtle peptides) extracted from Chinese pond turtles are bioactive peptides with multiple physiological functions: The amino acids in turtle protein peptides (such as arginine and taurine) can promote energy metabolism, enhance muscle endurance and recovery ability, improve the function of the nervous system, and relieve mental fatigue; turtle protein peptides are rich in various amino acids and bioactive substances, which can stimulate the activity of immune cells and enhance the body's immune function; the polypeptide chains in turtle protein peptides contain antioxidant groups, which can neutralize free radicals, protect cell membranes and DNA from oxidative damage, and promote collagen synthesis, helping to maintain skin elasticity and luster; turtle protein peptides are rich in collagen and various amino acids, which can promote the repair and regeneration of damaged tissues, making them particularly suitable for postoperative recovery, sports injury repair, and skin health maintenance; the collagen peptides in turtle protein peptides can promote the proliferation and repair of chondrocytes, increase the secretion of synovial fluid, and improve joint flexibility and comfort; turtle protein peptides are small molecule peptides, easily absorbed and utilized by the human body, and can improve the bioavailability of other nutrients in oral liquids.

[0005] Enzymatic hydrolysis of traditional Chinese medicine (TCM) that is both food and medicine involves using bio-enzyme technology to process the medicinal materials, breaking down large molecules into smaller active ingredients, thereby improving their bioavailability and efficacy. Enzymatic hydrolysis of TCM has significant advantages in anti-fatigue oral liquids. It has the effects of improving energy metabolism, enhancing immunity, anti-oxidation, improving blood circulation, and regulating the nervous system. Its highly efficient absorption and natural safety make it an important component in anti-fatigue oral liquids, suitable for a variety of people.

[0006] Currently, there is limited research on the preparation of oral liquids with excellent anti-fatigue effects by combining turtle protein peptides (small molecule turtle peptides) with enzymatically hydrolyzed medicinal and edible materials obtained through enzymatic hydrolysis and freeze-drying; therefore, relevant technical research is needed to fill the gap in this field. Summary of the Invention

[0007] To address the aforementioned technical problems, this invention specifically relates to a turtle protein peptide anti-fatigue oral liquid based on an enzymatic hydrolysis freeze-drying process and its preparation method.

[0008] This invention provides a method for preparing a turtle protein peptide anti-fatigue oral liquid based on an enzymatic hydrolysis and freeze-drying process, comprising the following steps:

[0009] Step 1: Preparation of enzymatic hydrolysate of medicinal and edible materials: Raw materials include: Rehmannia glutinosa, Astragalus membranaceus, Polygonatum sibiricum, Lycium barbarum, Citrus medica, Panax ginseng, Cistanche deserticola, and Rosa rugosa; enzymatic hydrolysis and freeze-drying process is used to prepare enzymatic hydrolysate of medicinal and edible materials;

[0010] Step 2: Prepare small molecule turtle peptides, sweeteners, acidulants, vitamins, and water;

[0011] Step 3: Mix the food-medicine homologous enzymatic hydrolysate, small molecule turtle peptide, sweetener, acidulant, vitamins and water, filter, sterilize, and fill to obtain the oral liquid.

[0012] Preferably, the dosage is as follows, by weight: 20-100 parts of enzymatically hydrolyzed material of medicinal and edible origin, 5-50 parts of small molecule turtle peptide, 0.1-10 parts of sweetener, 0.1-10 parts of acidulant, 0.001-0.1 parts of vitamin, and 800-1200 parts of water.

[0013] Preferably, the amount of enzymatically hydrolyzed material that is both food and medicine can also be a value or range of 22, 25, 28, 30, 35, 40, 45, 50, 60, 70, 80, or 90 parts.

[0014] Preferably, the enzymatic hydrolysis freeze-drying process includes enzymatic hydrolysis of Rehmannia glutinosa, Astragalus membranaceus, Polygonatum sibiricum, Lycium barbarum, Citrus medica, Panax ginseng, Cistanche deserticola and Rosa rugosa using a compound enzyme, followed by inoculation with fermentation bacteria for anaerobic fermentation and freeze-drying.

[0015] Preferably, when preparing the enzymatic hydrolysate of medicinal and edible materials, the raw materials are measured by mass parts as follows: 0.5-2 parts Rehmannia glutinosa, 0.5-2 parts Astragalus membranaceus, 0.5-2 parts Polygonatum sibiricum, 0.5-1.8 parts Lycium barbarum, 0.5-2.2 parts Citrus medica, 0.6-2.5 parts Panax ginseng, 0.6-2.4 parts Cistanche deserticola, and 0.5-2 parts Prickly pear.

[0016] Preferably, when preparing enzymatically hydrolyzed materials that are both food and medicine, the raw materials are crushed, boiled with 10-100 times their weight of water, cooled, and then enzymatically hydrolyzed.

[0017] Preferably, the complex enzyme comprises ginger protease and nattokinase in a mass ratio of 1.5-3:1. The enzyme parameters of ginger protease are 100-500 u / mg, and the enzyme parameters of nattokinase are 10000-50000 fu / g.

[0018] Preferably, the fermentation bacteria include *Lactobacillus casei* CI CC 22709 and *Brachystomata buddingus* ACCC 30144 in a ratio of 1:0.3-0.6.

[0019] Preferably, the steps for preparing enzymatically hydrolyzed materials that are both food and medicine include:

[0020] S1: Prepare the raw materials according to the following weight proportions: 0.5-2 parts Rehmannia glutinosa, 0.5-2 parts Astragalus membranaceus, 0.5-2 parts Polygonatum sibiricum, 0.5-1.8 parts Lycium barbarum, 0.5-2.2 parts Citrus medica, 0.6-2.5 parts Panax ginseng, 0.6-2.4 parts Cistanche deserticola, and 0.5-2 parts Rosa rugosa;

[0021] S2: Crush the above raw materials to obtain pulverized material, add 10-100 times the mass of water to the pulverized material, boil and then cool to obtain material S2;

[0022] S3: Add a complex enzyme composed of ginger protease and nattokinase in a mass ratio of 1.5-3:1 to the S1 material. The mass of the complex enzyme is 0.1-3% of the mass of the S1 material. Stir at 30-60℃ for 2-5 hours to hydrolyze the enzyme and inactivate it to obtain the S3 material.

[0023] S4: Add 1-4% glucose by weight of S3 material to S3 material, stir evenly to obtain intermediate material; then inoculate with a fermentation strain composed of *Lactobacillus casei* CICC 22709 and *Bacillus buddingus* ACCC 30144 at a ratio of 1:0.3-0.6. The amount of fermentation strain in the intermediate material is (2-7)×10 9 cfu / g; anaerobic fermentation at 25-40℃ for 5-10 days, sterilization, to obtain S4 material;

[0024] S5: The material from S4 is filtered, concentrated, and freeze-dried to obtain the enzymatically hydrolyzed material that is both medicinal and edible.

[0025] Preferably, the amount of small molecule turtle peptide can also be 6, 7, 9, 10, 15, 20, 25, 30, 35, 40, or 45 parts or a range thereof.

[0026] Preferably, the small molecule turtle peptide can be obtained commercially or prepared using conventional preparation processes in the art. For example, it can be obtained by crushing, soaking in acid or alkali, grinding, hydrolyzing with a protease, centrifuging, and freeze-drying the shell of a Chinese pond turtle. The protease can be Novozymes Protamex 1.6.

[0027] Preferably, the sweetener includes at least one of fructooligosaccharides, mogrosides, and xylitol. Its dosage can also be a value or range of 0.2, 0.3, 0.5, 1, 2, 5, 7, 8, or 9 parts.

[0028] Preferably, the acidulant includes at least one of malic acid, citric acid, and lactic acid. Its dosage may also be a value or range of 0.2, 0.3, 0.5, 1, 2, 5, 7, 8, or 9 parts.

[0029] Preferably, the vitamin includes at least one of vitamin B1, vitamin B2, vitamin B6, vitamin E, and vitamin C. The dosage may also be a value or range of 0.002, 0.005, 0.008, 0.01, 0.02, 0.05, 0.08, or 0.1 parts.

[0030] Preferably, the oral liquid may also contain selectively added chelating agents, preservatives, flavorings, colorings, viscosity control agents, and other ingredients, depending on functional requirements.

[0031] The present invention also provides a turtle protein peptide anti-fatigue oral liquid prepared according to the above preparation method based on an enzymatic hydrolysis freeze-drying process.

[0032] Compared to existing technologies, the advancement of this invention lies in:

[0033] This invention is the first to combine turtle protein peptides (small molecule turtle peptides) with enzymatically hydrolyzed medicinal and edible materials obtained through enzymatic hydrolysis and freeze-drying to prepare an anti-fatigue oral liquid. The small molecule turtle peptides can exert their bioactive peptide effects, enhancing muscle endurance and recovery ability, relieving fatigue, strengthening the body's immune function, and maintaining skin elasticity and luster. Other medicinal and edible raw materials, such as Rehmannia glutinosa, Astragalus membranaceus, Polygonatum sibiricum, Lycium barbarum, Citrus medica, Panax ginseng, Cistanche deserticola, and Rosa rugosa, can exert their respective medicinal effects, providing multiple amino acids, polysaccharides, flavonoids, minerals, vitamins, and other active ingredients, which have effects such as increasing resistance, anti-fatigue, sleep aid, and anti-oxidation. The synergistic effects of the various raw materials in the oral liquid give it excellent efficacy.

[0034] The oral liquid prepared by this invention has good low-temperature and high-temperature stability, and the product performance is stable.

[0035] The oral liquid obtained by this invention can significantly increase the effective swimming time of mice and has good anti-fatigue properties; it can significantly reduce the serum urea nitrogen content and increase the liver glycogen content of mice, which further verifies in principle that the oral liquid prepared by this invention has excellent anti-fatigue effects.

[0036] This invention uses dried pork floss and Buddha's hand as raw materials, which can further enhance the fatigue-inhibiting effect of oral liquid.

[0037] This invention utilizes a combination of ginger protease and nattokinase for enzymatic hydrolysis, which, compared to other enzymes, effectively enhances the anti-fatigue effect of the oral liquid. Furthermore, an oral liquid with a mass ratio of 1.5-3:1 yields an even better anti-fatigue effect.

[0038] This invention employs a co-fermentation process using *Lactobacillus casei* CI CC 22709 and *Bacillus buddingus* ACCC 30144, which enhances the anti-fatigue effect of the oral liquid, resulting in a better overall effect. When the ratio of *Lactobacillus casei* CI CC 22709 to *Bacillus buddingus* ACCC 30144 used in the fermentation is 1:0.3-0.6, the resulting oral liquid exhibits even better anti-fatigue effects.

[0039] The oral liquid of this invention has excellent effects, is easy to prepare, and has good application prospects. Detailed Implementation

[0040] The technical solution of the present invention will be clearly and completely described below with reference to the embodiments of the present invention. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. The following description of at least one exemplary embodiment is merely illustrative and is in no way intended to limit the present invention or its application or use. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.

[0041] Information on the raw materials used in the specific implementation method:

[0042] Monk fruit glycosides: Shaanxi Zelang Biotechnology Co., Ltd. Malic acid: L-malic acid, Shandong Jurong Bioengineering Co., Ltd. Ginger protease: 400u / mg, Shanghai Yimiao Chemical Technology Co., Ltd. Nattokinase: 50000fu / g, Shandong Aicai Biotechnology Co., Ltd. Bromelain: 300u / mg, Shanghai Yuanye Biotechnology Co., Ltd.

[0043] The preparation steps of small molecule turtle peptide are as follows:

[0044] (1) Dry and crush the shell of the grass turtle. Soak it in a 0.2 mol / L acetic acid solution with a concentration of 15 times the mass of the crushed material for 10 hours. Take it out, drain it and rinse it with clean water. Then soak it in a 0.1 mol / L NaOH aqueous solution with a concentration of 15 times the mass for 5 hours. Take it out, drain it and rinse it with clean water. Then add 30 times the amount of water, boil it and soak it for 0.5 hours. Take it out and drain it for later use.

[0045] (2) The product obtained in step (1) is ground by a grinder and sieved to obtain powder with a particle size of less than 0.1 mm. Then, 30 times the weight of water is added to the powder, and the complex protease Protamex (Novozymes Protamex 1.6) is added for enzymatic hydrolysis. The hydrolysis conditions are: pH = 7.0, hydrolysis temperature 42℃, hydrolysis time 4.5 h, enzyme dosage is 0.45% of the substrate powder mass, and during the hydrolysis process, the solution is electrolyzed for 70 s every 30 min. The pulse power supply voltage is 55 V and the frequency is 50 Hz to obtain the hydrolysis product.

[0046] (3) Inactivate the enzyme in the enzymatic hydrolysis product by water bath at 85°C for 10 min. Cool naturally to room temperature, centrifuge at 8000 r / min and 4°C for 0.5 hours, take the supernatant and freeze dry to a water content of 1.98 wt% to obtain turtle protein peptide powder.

[0047] I. Preparation of Oral Liquid

[0048] Oral liquid 1: The preparation steps are as follows:

[0049] Step 1: Preparation of enzymatic hydrolysate materials that are both medicinal and edible, the steps are as follows:

[0050] S1: Prepare the raw materials according to the following weight proportions: 1 part Rehmannia glutinosa, 1.2 parts Astragalus membranaceus, 1.2 parts Polygonatum sibiricum, 1.5 parts Lycium barbarum, 1.8 parts Citrus medica, 2 parts Panax ginseng, 2 parts Cistanche deserticola and 1.5 parts Prickly pear;

[0051] S2: Crush the above raw materials to obtain pulverized material, add 30 times the mass of water, boil, and then cool to room temperature to obtain material S2;

[0052] S3: Add a complex enzyme composed of ginger protease and nattokinase in a mass ratio of 2:1 to material S1, with the mass of the complex enzyme being 1.02% of the mass of material S1; stir at 50°C for 4.2 hours to hydrolyze the enzyme, inactivate the enzyme, and obtain material S3;

[0053] S4: Add 2.9% glucose (by weight of S3) to the S3 material and stir well to obtain an intermediate material; then inoculate with a fermentation strain composed of *Lactobacillus casei* CICC 22709 and *Cladosporium buddingii* ACCC 30144 at a ratio of 1:0.5. The amount of fermentation strain in the intermediate material is 6.0 × 10⁻⁶. 9 cfu / g; anaerobic fermentation at 35℃ for 8 days with stirring, followed by sterilization to obtain S4 material;

[0054] S5: The S4 material was filtered through a 0.22-micron pore size filter membrane, concentrated, and freeze-dried to a water content of 2.04 wt% to obtain the medicinal and edible homologous enzymatic hydrolysate.

[0055] Step 2: Prepare small molecule turtle peptides, sweeteners, acidulants, vitamins, and water;

[0056] Step 3: Mix 80 parts by weight of the enzymatically hydrolyzed material of medicinal and edible origin, 15 parts by weight of small molecule turtle peptide, 1 part by weight of sweetener mogroside, 1.5 parts by weight of acidifier malic acid, 0.037 parts by weight of vitamins (0.002 parts by weight of vitamin B1, 0.002 parts by weight of vitamin B2, 0.003 parts by weight of vitamin B6, 0.01 parts by weight of vitamin E, and 0.02 parts by weight of vitamin C) and 1000 parts by weight of water. Filter the mixture using a 0.22-micron pore size filter membrane, sterilize, and fill the container to obtain the oral liquid.

[0057] Oral liquid 2: The preparation steps are as follows:

[0058] Step 1: Preparation of enzymatic hydrolysate materials that are both medicinal and edible, the steps are as follows:

[0059] S1: Prepare the raw materials according to the following weight proportions: 2 parts Rehmannia glutinosa, 1.8 parts Astragalus membranaceus, 0.5 parts Polygonatum sibiricum, 0.5 parts Lycium barbarum, 1 part Citrus medica, 2.5 parts Panax ginseng, 2.2 parts Cistanche deserticola and 0.5 parts Prickly pear;

[0060] S2: Crush the above raw materials to obtain pulverized material, add 35 times the mass of water, boil, and then cool to room temperature to obtain material S2;

[0061] S3: Add a complex enzyme composed of ginger protease and nattokinase in a mass ratio of 3:1 to material S1. The mass of the complex enzyme is 1.15% of the mass of material S1. Stir at 45°C for 4 hours to hydrolyze the enzyme and inactivate it to obtain material S3.

[0062] S4: Add 2.5% glucose (by weight of S3) to the S3 material and stir well to obtain an intermediate material; then inoculate with a fermentation strain composed of *Lactobacillus casei* CICC 22709 and *Brevipedia buddingis* ACCC 30144 at a ratio of 1:0.3. The amount of fermentation strain in the intermediate material is 6.2 × 10⁻⁶. 9 cfu / g; anaerobic fermentation at 33℃ for 7 days with stirring, followed by sterilization to obtain S4 material;

[0063] S5: The S4 material was filtered through a 0.22-micron pore size filter membrane, concentrated, and freeze-dried to a water content of 2.12 wt% to obtain the medicinal and edible homologous enzymatic hydrolysate.

[0064] Step 2: Prepare small molecule turtle peptides, sweeteners, acidulants, vitamins, and water;

[0065] Step 3: Mix 63.5 parts by weight of the enzymatically hydrolyzed material of medicinal and edible origin, 12.5 parts by weight of small molecule turtle peptide, 0.8 parts by weight of sweetener mogroside, 1.15 parts by weight of acidifier malic acid, 0.0295 parts by weight of vitamins (0.0015 parts by weight of vitamin B1, 0.0015 parts by weight of vitamin B2, 0.0025 parts by weight of vitamin B6, 0.008 parts by weight of vitamin E, and 0.016 parts by weight of vitamin C) and 800 parts by weight of water. Filter the mixture through a 0.22-micron pore size filter membrane, sterilize, and fill the container to obtain the oral liquid.

[0066] Oral liquid 3:

[0067] The difference between S1 and oral liquid 1 is as follows: S1: Prepare raw materials according to the following weight parts: 1 part Rehmannia glutinosa, 1.2 parts Astragalus membranaceus, 1.2 parts Polygonatum sibiricum, 1.5 parts Lycium barbarum, 2 parts Ginseng and 1.5 parts Prickly pear.

[0068] Oral liquid 4:

[0069] The difference between S1 and oral liquid 1 is as follows: S3: Add a compound enzyme composed of ginger protease and natto kinase in a mass ratio of 2:1 to the S1 material. The mass of the compound enzyme is 1.5% of the mass of the S1 material. Stir at 50℃ for 4.2 hours to inactivate the enzyme and obtain the S3 material.

[0070] Oral liquid 5:

[0071] The difference between S1 and oral liquid 1 is as follows: S3: Add a compound enzyme composed of ginger protease and natto kinase in a mass ratio of 0.2:1 to the S1 material. The mass of the compound enzyme is 1.02% of the mass of the S1 material. Stir at 50℃ for 4.2 hours to hydrolyze the enzyme and inactivate it to obtain the S3 material.

[0072] Oral liquid 6:

[0073] The difference between S1 and oral liquid 1 is as follows: S3: Add a compound enzyme composed of ginger protease and natto kinase in a mass ratio of 10:1 to the S1 material. The mass of the compound enzyme is 1.02% of the mass of the S1 material. Stir at 50℃ for 4.2 hours to inactivate the enzyme and obtain the S3 material.

[0074] Oral liquid 7:

[0075] The difference from oral liquid 1 is as follows: S4: Add 2.9% glucose by weight of S3 material to S3 material, stir evenly to obtain intermediate material; then inoculate with Lactobacillus casei CI CC 22709 as fermentation bacteria, the amount of which in the intermediate material is 6.0 × 10⁻⁶. 9 cfu / g; anaerobic fermentation at 35℃ for 8 days with stirring, followed by sterilization to obtain S4 material.

[0076] Oral liquid 8:

[0077] The difference from oral liquid 1 is as follows: S4: Add 2.9% glucose by weight of S3 material to S3 material, stir evenly to obtain intermediate material; then inoculate with *Bacillus buddingus* ACCC 30144 as fermentation bacteria, the amount of which in the intermediate material is 6.0 × 10⁻⁶. 9 cfu / g; anaerobic fermentation at 35℃ for 8 days with stirring, followed by sterilization to obtain S4 material.

[0078] Oral liquid 9:

[0079] The difference from oral liquid 1 is as follows: S4: Add 2.9% glucose by weight of S3 material to S3 material, stir evenly to obtain intermediate material; then inoculate with a fermentation strain composed of Lactobacillus casei CICC 22709 and Cladosporium buddingii ACCC 30144 in a quantity ratio of 1:0.1. The amount of fermentation strain in the intermediate material is 6.0 × 10⁻⁶. 9 cfu / g; anaerobic fermentation at 35℃ for 8 days with stirring, followed by sterilization to obtain S4 material.

[0080] Oral liquid 10:

[0081] The difference from oral liquid 1 is as follows: S4: Add 2.9% glucose (by weight of S3 material) to S3 material, stir evenly to obtain intermediate material; then inoculate with a fermentation strain composed of *Lactobacillus casei* CICC 22709 and *Bacillus buddingus* ACCC30144 in a ratio of 1:3. The amount of fermentation strain in the intermediate material is 6.0 × 10⁻⁶. 9 cfu / g; anaerobic fermentation at 35℃ for 8 days with stirring, followed by sterilization to obtain S4 material.

[0082] II. Testing of Oral Liquids

[0083] 1. Stability

[0084] The above oral liquid was frozen at -10℃ for 7 days, then removed and allowed to naturally warm to room temperature. Its appearance was observed (whether there was discoloration or precipitation) to test its low-temperature stability. The oral liquid was then stirred in a 50℃ water bath for 12 hours, the water bath treatment was stopped, and it was allowed to cool naturally to room temperature. Its appearance was observed (whether there was discoloration or precipitation) to test its high-temperature stability. The test results are shown in Table 1.

[0085] Table 1: Stability

[0086]

[0087]

[0088] As shown in Table 1, the oral liquid prepared by this invention has good low-temperature and high-temperature stability, and the product performance is stable.

[0089] 2. Fatigue resistance test

[0090] Healthy male Kunming mice weighing 35±2g were selected as test subjects. After 3 days of acclimatization feeding, they were randomly divided into a control group and oral liquid groups 1-10, with 15 mice randomly assigned to each group based on their weight. Each group of mice underwent the three experimental tests described above after administration. Specifically, the oral liquid groups 1-10 were administered the oral liquid at a dose of 650mg / kg body weight via gavage once daily; the control group was administered an equal volume of physiological saline via gavage at the same frequency and method. During the gavage period, mice in each group had free access to food and water, and the administration continued for 35 days. Half an hour after the end of administration, the mice's weight-bearing swimming time was tested according to the method described in the 2023 edition of "Evaluation Procedures and Test Methods for Functional Evaluation of Health Foods," followed by testing of serum urea nitrogen and liver glycogen content. The test results are shown in Table 2.

[0091] Table 2: Weight-bearing swimming time, serum urea nitrogen content, and liver glycogen content

[0092]

[0093] The parameters in Table 2 were converted by difference, specifically by taking the absolute value of the difference between each group of indicators and the blank group. The results are shown in Table 3.

[0094] Table 3: Absolute values ​​of the differences in test indicators between each group and the blank group

[0095]

[0096]

[0097] Based on the results in Tables 2-3, it can be seen that the oral liquid obtained by the present invention can significantly increase the effective swimming time of mice and has good anti-fatigue properties; and compared with the blank group, it can significantly reduce the serum urea nitrogen content and increase the liver glycogen content of mice, which further verifies in principle that the oral liquid prepared by the present invention has excellent anti-fatigue effect.

[0098] Comparing oral liquids 1 and 3, it can be concluded that using pork floss and Buddha's hand as raw materials can further enhance the fatigue-inhibiting effect of the oral liquid.

[0099] Comparing oral liquids 1 and 4, it can be seen that the present invention uses a combination of ginger protease and nattokinase for enzymatic hydrolysis, which can effectively enhance the anti-fatigue effect of the oral liquid compared with other enzymes.

[0100] Comparing oral solutions 1 and 5-6, it can be seen that the oral solution obtained without using the compound enzyme composed of ginger protease and nattokinase in the mass ratio of 1.5-3:1 as described in this invention is less effective than the combination with a mass ratio of 1.5-3:1. The two enzymes used in this invention, within this ratio range, can further enhance the anti-fatigue effect of the oral solution.

[0101] A comparison of oral solutions 1 and 7-8 shows that the co-fermentation of *Lactobacillus casei* CICC 22709 and *Bacillus buddingus* ACCC 30144 in this invention is more effective than using only one type of fermentation in oral solutions 7-8. The co-fermentation of both enhances the anti-fatigue effect of the oral solutions, resulting in better efficacy. Tests on oral solutions 1 and 9-10 show that when the ratio of *Lactobacillus casei* CICC 22709 to *Bacillus buddingus* ACCC 30144 used in fermentation is 1:0.3-0.6, the resulting oral solution exhibits a better anti-fatigue effect.

[0102] The above embodiments are only used to illustrate the technical solutions of the present invention, and are not intended to limit them. Although the invention has been described in detail with reference to the foregoing embodiments, those skilled in the art can still modify the technical solutions described in the foregoing embodiments or make equivalent substitutions for some of the technical features. However, these modifications or substitutions do not cause the essence of the corresponding technical solutions to deviate from the spirit and scope of the technical solutions claimed by the present invention.

Claims

1. A method for preparing a turtle protein peptide anti-fatigue oral liquid based on enzymatic hydrolysis and freeze-drying process, characterized in that, Includes the following steps: Step 1: Preparation of enzymatic hydrolysate of medicinal and edible materials: S1: Prepare raw materials according to the following weight parts: 2 parts Rehmannia glutinosa, 1.8 parts Astragalus membranaceus, 0.5 parts Polygonatum sibiricum, 0.5 parts Lycium barbarum, 1 part Citrus medica, 2.5 parts Panax ginseng, 2.2 parts Cistanche deserticola and 0.5 parts Prickly pear; S2: Crush the above raw materials to obtain pulverized material, add 10-100 times the mass of water to the pulverized material, boil and then cool to obtain material S2; S3: Add a complex enzyme composed of ginger protease and nattokinase in a mass ratio of 1.5-3:1 to the S1 material. The mass of the complex enzyme is 0.1-3% of the mass of the S1 material. Stir at 30-60℃ for 2-5 hours to hydrolyze the enzyme and inactivate it to obtain the S3 material. S4: Add 1-4% glucose by weight of S3 material to S3 material, stir evenly to obtain intermediate material; then inoculate with a fermentation strain composed of *Lactobacillus casei* CICC 22709 and *Bacillus buddingus* ACCC 30144 at a ratio of 1:0.3-0.

6. The amount of fermentation strain in the intermediate material is (2-7)×10 9 cfu / g; anaerobic fermentation at 25-40℃ for 5-10 days, sterilization, to obtain S4 material; S5: The material from S4 is filtered, concentrated, and freeze-dried to obtain the enzymatically hydrolyzed material that is both medicinal and edible. Step 2: Prepare small molecule turtle peptides, sweeteners, acidulants, vitamins, and water; Step 3: Mix 20-100 parts by weight of the enzymatically hydrolyzed material of medicinal and edible origin, 5-50 parts by weight of small molecule turtle peptide, 0.1-10 parts by weight of sweetener, 0.1-10 parts by weight of acidifier, 0.001-0.1 parts by weight of vitamin and 800-1200 parts by weight, filter, sterilize and fill into the container to obtain the oral liquid.

2. The preparation method according to claim 1, characterized in that, Small molecule turtle peptides are obtained by crushing, soaking in acid solution, soaking in alkali solution, grinding, enzymatic hydrolysis, centrifugation and freeze-drying of Chinese pond turtle shell.

3. The preparation method according to claim 1, characterized in that, Sweeteners include at least one of fructooligosaccharides, mogrosides, and xylitol.

4. The preparation method according to claim 1, characterized in that, Acidulants include at least one of malic acid, citric acid, and lactic acid.

5. The preparation method according to claim 1, characterized in that, Vitamins include at least one of vitamin B1, vitamin B2, vitamin B6, vitamin E, and vitamin C.

6. A turtle protein peptide anti-fatigue oral liquid prepared by the preparation method according to any one of claims 1-5 based on enzymatic hydrolysis and freeze-drying process.

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