Wood frog antifreeze peptide as well as extraction method and application thereof

Abstract

The invention relates to wood frog antifreeze peptide and an extraction method and application thereof, and belongs to the field of biotechnology application. The method comprises the following steps: crushing wood frogs or wood frog tissues, pulping, adding protease for enzymolysis, deactivating enzyme, centrifuging, collecting supernate, passing through an ultrafiltration membrane with the molecular weight cutoff of 3-5kDa, collecting filtrate, and drying to obtain a wood frog antifreeze peptide crude product, thereby further obtaining the purified wood frog antifreeze peptide. The compound has the advantages that the freezing point of a solution can be nondependently reduced, a remarkable heat stagnation effect is shown, formation and growth of ice crystals can be actively inhibited, and the compound is used for preparing food or health-care food for improving the anti-freezing capability and anti-fatigue capability of an organism in a cold environment. The compound is used for preparing a protective agent for protecting biological tissues from physical and chemical damages in freezing, freeze thawing and cold-chain transportation processes, and is used for preparing a plant anti-frost agent, and tissue experiments show that the compound has an excellent protection effect on biological tissue structures, is superior to a traditional anti-freezing agent, and has huge potential in the fields of food preservation and biological sample low-temperature storage.

Description

Technical Field

[0001] This invention belongs to the field of biotechnology applications, specifically relating to forest frog antifreeze peptides, their extraction methods, and applications. Background Technology

[0002] Low-temperature freezing is widespread in nature and human activities, posing a severe challenge to organisms, industrial and agricultural production, and daily life. In the biomedical field, frostbite (or non-freezing cold injury) is a common local inflammatory skin disease caused by cold exposure, typically manifesting as erythema, swelling, itching, and blisters at the exposed site. Its pathological basis lies in the local blood circulation disorders and tissue inflammation caused by low temperatures. Existing clinical interventions, such as vasodilators or topical camphor preparations, often have limitations in efficacy, short-lived effects, or skin irritation. In the consumer goods and industrial sectors, low-temperature environments also cause numerous problems. For example, outdoor workers or people in high-altitude and cold regions face the risk of frostbite and chapped skin; while the surfaces of medical devices, precision instruments, and vehicles are prone to performance degradation, malfunctions, and even safety hazards due to icing. Current industrial de-icing technologies mostly rely on high-energy heating or corrosive chemical salts, the negative impacts of which on the environment and the equipment itself cannot be ignored.

[0003] The core objective of "antifreeze" technology is to block or delay freezing, thereby mitigating or preventing various types of damage caused by freezing. An ideal antifreeze strategy should combine high efficiency with biocompatibility. In the pharmaceutical field, developing topical formulations that improve local microcirculation, inhibit inflammatory responses, and protect tissue cells from low-temperature damage will be an ideal solution for preventing and treating frostbite. In skincare, there is an urgent need for active ingredients that can provide long-lasting protection for skin exposed to cold environments, preventing moisture loss and low-temperature stimulation. In industry, exploring green, low-corrosion, and highly effective freeze inhibitors is also an important research direction in materials science and engineering.

[0004] Organisms in nature with exceptional cold resistance provide valuable resources for addressing the aforementioned need for cryogenic protection. The forest frog of Changbai Mountain in my country possesses a unique cold-region survival strategy. This species needs to hibernate for 6 to 7 months each year under snow and ice, yet its tissues and cells effectively avoid freezing damage under these extreme conditions. This unique cryogenic ability is attributed to a special class of biomolecules synthesized during its long evolutionary process—forest frog antifreeze peptides. These antifreeze peptides can lower the freezing point of body fluids and inhibit ice crystal growth and recrystallization through non-colligative mechanisms, thus providing crucial cryogenic protection for the forest frog at the molecular level. However, to date, the development and application of forest frog antifreeze peptides have not been reported. Their enormous application potential, especially their transformative value in developing high-value-added pharmaceuticals, skincare products, food, and industrial protective products, remains largely untapped, representing a significant technological gap. Summary of the Invention

[0005] This invention provides a forest frog antifreeze peptide, its extraction method, and its application. The purpose is to overcome the limitations of existing technologies, make full use of the special biological resource of the forest frog, and deeply develop the application of forest frog antifreeze peptide in the preparation of antifreeze drugs, food, skin care products, and industrial protective products, so as to meet the urgent antifreeze needs of multiple fields.

[0006] The technical solution adopted in this invention is that a forest frog antifreeze peptide is obtained by the following steps:

[0007] (1) The frog or frog tissue is crushed and pulped to obtain a frog homogenate;

[0008] (2) Adjust the pH of the homogenate to 6.0-7.5, add 1.0%-3.0% of the substrate mass of protease, and enzymatically hydrolyze at 45-55℃ for 3-6 hours;

[0009] (3) Heat the enzyme hydrolysate to 95-100℃ and keep it for 10-15 minutes to inactivate the enzyme. After cooling, centrifuge at 4℃ and 8000-12000 rpm and collect the supernatant.

[0010] (4) Pass the supernatant through an ultrafiltration membrane with a molecular weight cutoff of 3kDa-5kDa, collect the filtrate and dry it to obtain crude antifreeze peptides from the forest frog;

[0011] (5) Prepare the crude product into a solution and load it onto a Sephadex G-15 gel chromatography column. Use purified water or 0.1 mol / L acetic acid solution as the eluent and control the flow rate at 0.5-1.0 mL / min. Collect the main peak component with a relative molecular mass concentrated in the 500-2000 Da range from the elution curve. After drying, obtain purified forest frog antifreeze peptide.

[0012] The relative molecular mass distribution of the forest frog antifreeze peptide of the present invention is between 180 Da and 2000 Da; and the thermal hysteresis activity of the forest frog antifreeze peptide is not less than 0.3℃ at a concentration of 10 mg / mL.

[0013] The frog tissue mentioned in step (1) of this invention is selected from one or a combination of frog eggs, frog skin, frog meat, frog bones, frog ova, or whole frog.

[0014] The protease mentioned in step (2) of this invention is composed of papain and flavor protease in a mass ratio of 1:1 to 3:1; or trypsin and neutral protease.

[0015] In step (4) of this invention, before ultrafiltration separation, the supernatant is pre-filtered through an ultrafiltration membrane with a molecular weight cutoff of 10 kDa, the cutoff liquid is discarded, and the filtrate is collected for subsequent operations.

[0016] A method for extracting antifreeze peptides from the forest frog includes the following steps:

[0017] (1) The frog or frog tissue is crushed and pulped to obtain a frog homogenate;

[0018] (2) Adjust the pH of the homogenate to 6.0-7.5, add 1.0%-3.0% of the substrate mass of protease, and enzymatically hydrolyze at 45-55℃ for 3-6 hours;

[0019] (3) Heat the enzyme hydrolysate to 95-100℃ and keep it for 10-15 minutes to inactivate the enzyme. After cooling, centrifuge at 4℃ and 8000-12000 rpm and collect the supernatant.

[0020] (4) Pass the supernatant through an ultrafiltration membrane with a molecular weight cutoff of 3kDa-5kDa, collect the filtrate and dry it to obtain crude antifreeze peptides from the forest frog;

[0021] (5) Prepare the crude product into a solution and load it onto a Sephadex G-15 gel chromatography column. Use purified water or 0.1 mol / L acetic acid solution as the eluent and control the flow rate at 0.5-1.0 mL / min. Collect the main peak component with a relative molecular mass concentrated in the 500-2000 Da range from the elution curve. After drying, obtain purified forest frog antifreeze peptide.

[0022] The application of the frog antifreeze peptide described in this invention in the preparation of cosmetics or topical skin preparations for the prevention or treatment of frostbite and chapped skin.

[0023] The application of the frog antifreeze peptide described in this invention in the preparation of food cryoprotectants, particularly for improving the texture and water-holding capacity of meat products or fruits and vegetables during storage.

[0024] The application of the forest frog antifreeze peptide described in this invention in the preparation of food or medicine for improving the body's cold resistance or relieving low-temperature exercise fatigue.

[0025] An antifreeze composition comprising: (a) an effective amount of forest frog antifreeze peptides; and (b) one or more pharmaceutically or cosmetically acceptable excipients selected from at least one of hyaluronic acid, glycerin, propylene glycol, trehalose, and ceramides.

[0026] The composition is a topical skin preparation, a health food, or an antifreeze spray for use on surfaces; wherein the forest frog antifreeze peptide in the composition is 0.1% to 20.0% by mass.

[0027] The advantages of this invention are:

[0028] The *Rana camara* antifreeze peptides provided by this invention possess one or more of the following excellent biophysical activities: They can noncollinearly lower the freezing point of solutions and exhibit significant thermal hysteresis; they can actively inhibit the formation and growth of ice crystals, rather than solely relying on concentration effects; they can effectively inhibit ice crystal growth and transform the ice crystal morphology from sharp, easily damaging large hexagonal shapes to rounded, less damaging needle-like or dendritic shapes; under low-temperature conditions, they can significantly protect the activity of bioactive macromolecules (such as catalase) and reduce oxidative stress damage caused by low temperatures; they can significantly reduce the frostbite area of ​​skin tissue and lower the levels of inflammatory factors (IL-6, TNF-α); in isolated tissues, they can effectively protect the integrity of muscle fiber structure and reduce dripping water loss during freeze-thaw cycles.

[0029] This invention reveals the broad application potential of forest frog antifreeze peptides in multiple fields: for the preparation of drugs or skincare products to prevent and / or treat frostbite of skin tissues; for the preparation of foods or health foods to enhance the body's resistance to freezing and fatigue in cold environments; for the preparation of protective agents to protect biological tissues (including muscle tissue, fruits, etc.) from physical and chemical damage during freezing, freeze-thaw cycles, and cold chain transportation; and for the preparation of plant antifrost agents, which are sprayed before frost to protect plant flowers and fruits from frost damage.

[0030] Through systematic research, this invention confirms that the forest frog antifreeze peptide possesses multiple antifreeze effects. It exhibits significant thermal hysteresis activity and ice crystal morphology inhibition capabilities, effectively mitigating ice crystal damage at a physical level. Furthermore, it effectively protects catalase activity, increasing its activity retention rate by approximately 32%, thus alleviating oxidative stress. Animal experiments show that the forest frog antifreeze peptide can efficiently prevent frostbite with an inhibition rate of 76.2%, significantly reducing inflammatory factor levels, demonstrating promising applications in the prevention and treatment of frostbite. Oral administration can systematically enhance the body's cold resistance, significantly prolong the time spent swimming in cold water at exhaustion, and improve metabolic indicators, providing a basis for the development of antifreeze functional foods. Tissue experiments demonstrate its superior protective effect on biological tissue structures compared to traditional antifreeze agents, showing great potential in food preservation and low-temperature storage of biological samples. Attached Figure Description

[0031] Figure 1 These are photographs of mouse skin affected by frostbite in this invention.

[0032] Figure 2 These are pathological sections of mouse skin frostbite experimentally presented in this invention;

[0033] Figure 3 This is a graph showing the IL-6 test results in the mouse skin frostbite experiment of this invention;

[0034] Figure 4 This is a graph showing the TNF-α test results in the mouse skin frostbite experiment of this invention;

[0035] Figure 5This is a differential scanning lithography result of the forest frog antifreeze peptide of the present invention;

[0036] Figure 6 This is a diagram showing the experimental results of the forest frog antifreeze peptide ice crystals of this invention;

[0037] Figure 7 This is a diagram showing the experimental results of the protection of animal tissues by the frog antifreeze peptide of this invention. Detailed Implementation

[0038] Example 1: Extraction of antifreeze peptides from forest frogs

[0039] (1) Take 1 kg of whole forest frogs, wash them and then crush them into a homogenate;

[0040] (2) Add purified water to the homogenate at a mass-to-volume ratio of 1:20 and mix well. Adjust the pH to 7.0 using a dilute alkaline solution. Add 2.0% of the total mass of the forest frog as neutral protease and heat at 50°C. o Enzymatic hydrolysis at constant temperature for 4 hours at C;

[0041] (3) After enzymatic hydrolysis, heat the solution to 95°C. o Inactivate enzymes at C for 10 minutes, then cool and incubate at 4°C. o C. Centrifuge at 10000 rpm for 30 minutes and collect the supernatant;

[0042] (4) Pass the supernatant through an ultrafiltration membrane with a molecular weight cutoff of 5 kDa, collect the filtrate, and freeze-dry the filtrate to obtain crude antifreeze peptides from the forest frog.

[0043] (5) The crude product was prepared into a solution and loaded onto a Sephadex G-15 gel chromatography column using purified water as the eluent. The flow rate was controlled at 0.8 mL / min. The main peak fraction with a relative molecular mass concentrated between 500 Da and 2000 Da in the elution curve was accurately collected. After drying, purified *Rana camara* antifreeze peptide powder was obtained. The thermal hysteresis activity of this peptide was determined to be 0.6%. o C.

[0044] Example 2: Extraction of antifreeze peptides from frog eggs

[0045] (1) Take 200g of frog eggs and crush them;

[0046] (2) Add purified water at a material-to-liquid ratio of 1:15, and adjust the pH to 6.0 using HCl solution; add papain and flavor protease complex enzyme (mass ratio 1:2) at 1.0% of the total mass of raw materials, and heat at 55°C. o Enzymatic hydrolysis at constant temperature for 3 hours at C;

[0047] (3) After the enzymatic hydrolysis is complete, heat the solution to 98°C. o Enzyme inactivation at C for 12 minutes, followed by cooling and then incubation at 4°C. oC. Centrifuge at 8000 rpm for 30 minutes and collect the supernatant;

[0048] (4) Pass the supernatant through a 5kDa ultrafiltration membrane system, collect the filtrate, and spray dry it to obtain 20.5g of crude product;

[0049] (5) The crude product was purified by Sephadex G-15 gel chromatography using purified water as the eluent at a flow rate of 0.5 mL / min. 12.2 g of *Rana camara* antifreeze peptides (500 Da–2000 Da) were collected. Thermal hysteresis activity was 0.55. o C.

[0050] Example 3: Extraction of antifreeze peptides from frog skin

[0051] (1) Take 200g of frog skin and grind it thoroughly into a pulp;

[0052] (2) Add purified water to the homogenate at a material-to-liquid ratio of 1:20, adjust the pH to 7.5 with NaOH solution, add trypsin and neutral protease complex enzyme (mass ratio 1:1) accounting for 2.5% of the mass of frog skin, and enzymatically hydrolyze at 45℃ for 6 hours.

[0053] (3) After the enzyme hydrolysate is inactivated at 100℃ for 15 minutes and centrifuged, the supernatant is collected. The supernatant is first passed through a 10kDa pre-ultrafiltration membrane, the retentate is discarded, and the filtrate is collected.

[0054] (4) The filtrate was then passed through a 3 kDa ultrafiltration membrane, and the retentate was collected and dried.

[0055] (5) After dissolving the dried crude *Rana camara* antifreeze peptides, the product was purified by Sephadex G-15 gel chromatography using 0.1 mol acetic acid solution as the eluent and a flow rate controlled at 1.0 mL / min. 5.6 g of *Rana camara* antifreeze peptides in the range of 500 Da to 2000 Da were collected, with a thermal hysteresis activity of 0.5%. o C.

[0056] Example 4: Extraction of antifreeze peptides from a mixture of frog meat and bones

[0057] (1) Take 150g of a mixture of frog meat and frog bone (mass ratio 2:1) and homogenize it using a homogenizer;

[0058] (2) Mix the homogenate with 1500mL of purified water (material-to-liquid ratio 1:10), stir in a 45℃ water bath, maintain the pH at 7.5 with phosphate buffer, add 3.0g of trypsin (addition amount is 2% w / w), and stir at 45℃ for 5 hours for enzymatic hydrolysis.

[0059] (3) After the enzymatic hydrolysis is completed, the mixture is placed in a boiling water bath and heated for 10 minutes to inactivate the enzyme. Then, the mixture is transferred to a centrifuge tube and centrifuged at 4°C and 12,000 rpm for 30 minutes.

[0060] (4) Pass the supernatant through an ultrafiltration membrane system with a molecular weight cutoff of 4 kDa and collect the filtrate;

[0061] (5) The filtrate was loaded onto a Sephadex G-15 gel chromatography column and eluted with ultrapure water at a flow rate of 0.7 mL / min. The main peak fraction with a molecular weight between 500 Da and 2000 Da was collected, dried, and 4.2 g of purified peptide powder was obtained with a thermal hysteresis activity of 0.52. o C.

[0062] Example 5: Experiment on the prevention / treatment of frostbite in mouse skin tissue by frog antifreeze peptides.

[0063] After anesthesia, the backs of mice were shaved. A 1.5cm diameter iron rod was immersed in liquid nitrogen for approximately 15 minutes to reach -196°C. After removal, the rod was placed directly onto the mouse skin for 8 seconds to establish the frostbite model. This experiment included three groups of mice: a control group, a model group, and a RDAFP (rfrog antifreeze peptide) group, with 10 mice in each group (half male and half female). In the control group, only hair removal cream was used to shave the backs of the mice; no other treatments were given. In the model group, hair removal cream was used to shave the backs of the mice, and a frostbite model was established using the above method. In the treatment group, hair removal cream was used to shave the backs of the mice, and 0.5mL of RDAFP solution (0.04g / mL) was applied topically to the exposed skin, allowing approximately 20 minutes for absorption. Then, the frostbite model was established in the mice using the above method. Ten days later, H&E staining was performed on the mouse skin sections to observe the degree of damage, and IL-6 and TNF-α levels were measured to assess inflammation at the frostbite site. The results showed that the antifreeze peptides from the forest frog could significantly reduce the area of ​​frostbite caused by low temperature (inhibition rate reached 76.2%). Figure 1 Appendix Figure 2 ), and reduce tissue inflammatory response (see appendix) Figure 3 Appendix Figure 4 This indicates that it has clear application potential in products such as antifreeze ointments and antifreeze face creams.

[0064] Table 1 Results of the experiment on the prevention of frostbite in mice by frog antifreeze peptides.

[0065] Group Frostbite area (mm²) Histopathological score (0-4 points) Degree of inflammatory cell infiltration control group 0 0 none Model group Larger (approximately 94.4 mm²) 4 large amount Antifreeze peptides <![CDATA[Smaller (about 9.3 mm 2 )]]> 1 small amount

[0066] Note: **p<0.01 (compared to the control group). Histopathological scoring criteria: 0 = no damage, 4 = full-thickness necrosis.

[0067] Example 6: Experiment on the effect of frog antifreeze peptides on the antifreeze ability of mice

[0068] Forty healthy ICR mice were randomly divided into four groups (n=10): a control group (saline), a low-dose group (100 mg / kg antifreeze peptide), a medium-dose group (200 mg / kg antifreeze peptide), and a high-dose group (400 mg / kg antifreeze peptide). After four weeks of continuous gavage, mice underwent a weight-bearing swimming test (5% of body weight load) in cold water at a temperature of 4°C. The time to exhaustion was recorded, and serum lactate (LA), blood urea nitrogen (BUN), and creatine kinase (CK) levels were measured. The results showed that the *Rana camara* antifreeze peptide significantly prolonged the time to exhaustion in cold water (79.1% increase in the medium-dose group) and significantly reduced post-exercise levels of blood lactate, BUN, and creatine kinase. This indicates that the *Rana camara* antifreeze peptide can improve physiological functions under cold conditions and can be used to prepare antifreeze foods.

[0069] Table 2. Experimental results on the improvement of the antifreeze ability of mice by frog antifreeze peptides.

[0070] Group Time to exhaustion (min) Serum LA (mmol / L) Serum BUN (mmol / L) Serum CK (U / L) control group 39.6±6.1 8.8±0.9 13.3±1.2 464±38 low-dose group 50.7±4.3* 7.3±0.7* 11.1±1.1* 388±22* medium dose group 66.2±3.8** 6.1±0.6** 9.2±0.9** 310±34** High-dose group 71.4±5.2** 5.7±0.5** 8.1±0.7** 296±21**

[0071] Note: **p<0.01, *p<0.5 (compared to the control group).

[0072] Example 7: Thermal hysteresis activity and ice crystal morphology experiment

[0073] The *Rana camara* antifreeze peptide was prepared into a 5 mg / mL solution using ultrapure water, with ultrapure water serving as a negative control. Differential scanning calorimetry (DSC) was used to test the samples. 10 μL of the sample solution was sealed in an aluminum crucible and cooled from 25 °C to -25 °C at a rate of 5 °C / min, then heated back to 25 °C at the same rate. The holding temperature and initial crystallization temperature of the sample were recorded, and the thermal hysteresis activity was calculated. The results are shown in the appendix. Figure 5 The freezing point of the *Rana camara* antifreeze peptide solution was significantly lowered, and it exhibited obvious thermal hysteresis activity (0.6℃). This indicates that the *Rana camara* antifreeze peptide can noncollinearly lower the freezing point of the solution and has excellent antifreeze activity.

[0074] Ice crystal morphology observation: The above samples were placed on the temperature-controlled stage of a cold-stage microscope and slowly cooled to -30℃. The ice crystal morphology was observed under the microscope. The results showed that the control group formed large and sharp hexagonal ice crystals, while the ice crystals formed by the frog antifreeze peptide group were significantly smaller in size and changed to rounded needle-like or dendritic shapes (see attached). Figure 6 This study demonstrates that the antifreeze peptides from the forest frog can effectively inhibit ice crystal growth and alter its morphology, thereby reducing the damage of ice crystals to animal tissues.

[0075] Example 8: Catalase Activity Protection Experiment

[0076] Low-temperature stress induces the production of reactive oxygen species in cells, leading to oxidative damage. This experiment indirectly demonstrates the cell protection ability of *Rana camara* antifreeze peptides by detecting their protective effect on catalase activity at low temperatures. *Rana camara* antifreeze peptides were dissolved in deionized water to prepare a 2 mg / mL solution. A certain amount of catalase was weighed and dissolved in 0.05 mol / L KH₂PO₄-NaOH buffer (pH=7.0) to a concentration of 0.2 mg / mL. The two solutions were mixed at a 1:2 volume ratio, and the initial enzyme activity was measured. A catalase solution without *Rana camara* antifreeze peptides was used as a control. The remaining mixture was then frozen at -20°C for 24 h, thawed at room temperature for 3 h, and this freeze-thaw cycle was repeated four times. The enzyme activity after the freeze-thaw cycle was then measured. Enzyme activity assay: Add 1 mL of water and 1 mL of 0.1 mol / L hydrogen peroxide solution to 1 mL of a mixed solution of catalase and frog antifreeze peptides, shake immediately, and measure the absorbance value (ΔA240) at 240 nm. Record the absorbance value every 1 min and measure the change in absorbance value over 10 min. Calculate the residual catalase activity based on the following:

[0077]

[0078]

[0079] Where Vt represents the total volume of the reaction solution; Vs represents the volume of the sample solution used for measurement; t represents the time from the addition of hydrogen peroxide solution to the last recording; and W represents the mass of the enzyme in the sample solution.

[0080] The results showed that compared with the control group without the addition of *Rana camara* antifreeze peptides (40.32±1.65), the experimental group with the addition of *Rana camara* antifreeze peptides had a significantly higher retention rate of catalase activity (72.41±2.53), which was concentration-dependent. This indicates that *Rana camara* antifreeze peptides can effectively alleviate the damage of low temperature to bioactive macromolecules and have the potential to resist oxidative stress.

[0081] Example 9: Animal Tissue Antifreeze Experiment

[0082] Fresh pork tenderloin was taken and cut into thin, uniform slices (2cm × 2cm × 0.5cm). The slices were randomly divided into three groups: a blank group (untreated pork pieces), a negative control group (pork pieces injected with physiological saline at a ratio of 1:10 (v / w), a positive control group (pork pieces injected with a commercial antifreeze agent (physiological saline containing 4% sucrose + 4% sorbitol) at a ratio of 1:10 (v / w), and an antifreeze peptide group (pork pieces injected with a frog antifreeze peptide solution (physiological saline containing 4% frog antifreeze peptide) at a ratio of 1:10 (v / w). All slices were then frozen at -20°C for 12 hours, followed by thawing at 4°C for 12 hours, constituting one cycle. Three cycles were performed. After thawing, the color and texture changes of the slices were observed, and the drip loss rate was measured. Paraffin sections were prepared from a portion of the meat samples, stained with H&E, and the integrity of the muscle fiber structure was observed under an optical microscope. After thawing, the tissue from the frog antifreeze peptide group remained bright red and retained good elasticity. Microscopic examination revealed intact muscle fibers and skin tissue structure, with highly regular muscle cell morphology, small and uniform intercellular spaces, and high integrity of muscle fiber structure. This indicates that the frog antifreeze peptide has a significant protective effect on pork muscle tissue during cryopreservation, and its antifreeze effect is superior to the negative control group. Compared with the positive control group, it also showed good antifreeze performance. (See appendix) Figure 7 Furthermore, the water droplet experiment showed that the forest frog antifreeze peptides can effectively alleviate water loss in pork during freezing. These results demonstrate that the forest frog antifreeze peptides can effectively protect biological tissues from low-temperature damage, providing a basis for their application in food, pharmaceuticals, and skincare products.

[0083] Example 10: Application in Fruit Freeze-Thaw Damage

[0084] This study verified the effectiveness of *Rhizophora stylosa* antifreeze peptides in protecting fruits (using strawberries as an example) from freeze-thaw damage. Fresh strawberries of uniform size and maturity were randomly divided into two groups. The control group was sprayed with deionized water. The treatment group was sprayed with a solution containing 1% (w / v) *Rhizophora stylosa* antifreeze peptides. After the surface liquid dried, all strawberries were frozen at -20℃ for 24 hours and then thawed naturally at room temperature. The shape of the strawberries after thawing was observed, including whether they collapsed and whether juice leaked out. After thawing, the strawberries in the control group softened severely, collapsed, and leaked a large amount of juice, resulting in a darker color. In contrast, the strawberries treated with *Rhizophora stylosa* antifreeze peptides maintained a relatively intact shape, leaked less juice, and had a brighter color. This indicates that *Rhizophora stylosa* antifreeze peptides can effectively maintain the integrity of fruit cell membranes and reduce the physical and quality damage caused by the freeze-thaw process.

[0085] Example 11 Preparation of Skin Antifreeze Care Composition

[0086] Forest frog antifreeze peptides 3% (w / w); glycerol 5%; sodium hyaluronate 0.5%; vitamin E 0.5%; allantoin 0.2%; conventional oil phase, aqueous phase and emulsion system; supplemented to 100%.

[0087] Example 12 Antifreeze Cream

[0088] Forest frog antifreeze peptides 3%; capsaicin 0.05%; glycerin + hyaluronic acid 8%; mannitol 5%; carbomer matrix; replenish to 100% to form a stable paste suitable for outdoor emergencies.

[0089] Example 13 Antifreeze spray for cold chain logistics

[0090] Forest frog antifreeze peptides 1.5%; trehalose 5%; polysorbate 0.5%; food-grade propylene glycol 10%; deionized water to 100%. This spray forms a transparent antifreeze protective film on metal, plastic and other surfaces, effectively delaying the formation and adhesion of ice crystals. The ingredients are biodegradable, non-corrosive, and food-grade, making it particularly suitable for use in food cold chains and precision instrument warehouses.

[0091] Example 14 Plant Frost Protection Spray

[0092] Frog antifreeze peptides 0.1%; abscisic acid 10 ppm; chitosan 0.2%; potassium dihydrogen phosphate 1%; water to 100%. Spraying before frost forms a protective layer on plant surfaces. Frog antifreeze peptides directly inhibit ice crystal formation, while abscisic acid and nutrients enhance the plant's cold resistance from within, providing double protection, reducing frost damage to flowers and fruits, and improving the yield and quality of agricultural products.

[0093] Example 15 Anti-freeze Repair Serum

[0094] The composition includes: 2% forest frog antifreeze peptides; 2% ectoine; 1% ceramide NP; 0.5% bisabolol; 5% squalane; and the essence matrix is ​​replenished to 100%. This composition constitutes an "environmental defense system." The forest frog antifreeze peptides address the physical damage caused by low temperatures, while ectoine and bisabolol counteract the resulting cellular stress and inflammation. Ceramide and squalane repair the skin barrier, working together to achieve deep antifreeze and repair.

[0095] Example 16 Antifreeze Oral Solution

[0096] Ingredients: 5.0g *Rana cambogia* antifreeze peptide powder; 20.0g isomaltooligosaccharide; 10.0g honey; 0.3g citric acid; 0.1g potassium sorbate; purified water to 100mL. Instructions: Add approximately 70mL of purified water to a mixing container and heat to 60-70℃. While stirring, add isomaltooligosaccharide and honey sequentially until completely dissolved. Cool the solution to below 40℃, add the *Rana cambogia* antifreeze peptide powder, and stir slowly until completely dissolved, avoiding excessive foaming. Add citric acid and potassium sorbate, and stir until completely dissolved. Add purified water to 100mL and stir well. Fill the prepared solution aseptically into 10mL oral liquid glass bottles and seal for sterilization.

[0097] Example 17 Antifreeze Repair Gel

[0098] 0.8% Carbomer 940 was dispersed in a portion of purified water and allowed to swell fully to form a transparent gel matrix. In another container, 20% *Rana camara* antifreeze peptide powder, 15% glycerol, and 2% ceramide NP were dissolved in the remaining aqueous phase. The aqueous active ingredient solution was slowly added to the gel matrix under continuous gentle stirring, ensuring uniform mixing. Finally, the pH was adjusted to 6.0–6.5 using triethanolamine to obtain a transparent, stable, high-concentration repair gel, and the volume was brought to 100.0%.

[0099] Example 18 Cold-resistant food

[0100] Mix 20.0% isomaltooligosaccharide with 18.0% honey (or maltitol) and heat to a syrupy consistency with appropriate viscosity. Then, thoroughly mix 35.0% pre-baked rolled oats, 15.0% chopped nuts, 10.0% forest frog antifreeze peptide powder, and 2.0% B vitamin complex. Pour the heated syrup into the dry powder mixture and stir quickly and thoroughly until all ingredients are evenly combined. Transfer the mixture to a pre-made mold, flatten and shape, and cut into approximately 50g energy bars after cooling.

[0101] Example 19 Plant-based antifrost agent

[0102] The composition consists of 5.0% *Rana camara* antifreeze peptide powder, 0.002% S-inducer (20 ppm), 0.5% chitosan oligosaccharide, 0.3% potassium dihydrogen phosphate, 0.05% organosilicon surfactant, and the remainder is purified water. During preparation, the solid components are dissolved sequentially in purified water. After complete dissolution, S-inducer is added, followed by the organosilicon surfactant, and the mixture is stirred at low speed until homogeneous before being bottled.

[0103] The above description is merely a preferred embodiment of the present invention, but the scope of protection of the present invention is not limited thereto. Any variations or substitutions that can be easily conceived by those skilled in the art within the technical scope disclosed in the present invention should be included within the scope of protection of the present invention. Therefore, the scope of protection of the present invention should be determined by the scope of the claims.

Claims

1. A Rana temporaria antifreeze peptide, characterized in that, is obtained by the following steps: (1) Pulverize Rana temporaria or Rana temporaria tissue to make pulp, and obtain Rana temporaria homogenate; (2) Adjust the pH of the homogenate to 6.0-7.5, add protease with a substrate mass of 1.0%-3.0%, and enzymatically hydrolyze at 45-55°C for 3-6 hours; (3) Heat the enzymatic hydrolysate to 95-100°C for 10-15 minutes to inactivate the enzyme, cool it, centrifuge it at 4°C and 8000-12000 rpm, and collect the supernatant; (4) Pass the supernatant through an ultrafiltration membrane with a molecular weight cut-off of 3kDa-5kDa, collect the filtrate, and dry it to obtain crude Rana temporaria antifreeze peptide; (5) Prepare a solution of the crude product, load it into a Sephadex G-15 gel chromatography column, use purified water or 0.1mol / L acetic acid solution as eluent, control the flow rate at 0.5-1.0mL / min, collect the main peak components with a relative molecular mass of 500-2000Da in the elution curve, and dry it to obtain purified Rana temporaria antifreeze peptide.

2. The Rana temporaria antifreeze peptide according to claim 1, characterized in that, The relative molecular mass of the Rana temporaria antifreeze peptide is distributed between 180Da and 2000Da; and the thermal hysteresis activity of the Rana temporaria antifreeze peptide at a concentration of 10mg / mL is not less than 0.3°C.

3. The Rana sylvatica antifreeze peptide of claim 1, wherein: The Rana temporaria tissue in step (1) is selected from one or a combination of several of Rana temporaria eggs, Rana temporaria skin, Rana temporaria meat, Rana temporaria bone, Rana temporaria ovum, or Rana temporaria whole frog.

4. The Rana sylvatica antifreeze peptide of claim 1, wherein: The protease in step (2) is composed of papain and flavourzyme with a mass ratio of 1:1 to 3:1; or trypsin, neutral protease.

5. The forest frog antifreeze peptide according to claim 1, characterized in that: Before ultrafiltration separation in step (4), the supernatant is first pre-filtered through an ultrafiltration membrane with a molecular weight cut-off of 10kDa, the retentate is discarded, and the filtrate is collected for subsequent operation.

6. The method of claim 1 to 5, wherein the Rana temporaria antifreeze peptide is extracted from the skin of Rana temporaria. including the following steps: (1) Pulverize Rana temporaria or Rana temporaria tissue to make pulp, and obtain Rana temporaria homogenate; (2) Adjust the pH of the homogenate to 6.0-7.5, add protease with a substrate mass of 1.0%-3.0%, and enzymatically hydrolyze at 45-55°C for 3-6 hours; (3) Heat the enzymatic hydrolysate to 95-100°C for 10-15 minutes to inactivate the enzyme, cool it, centrifuge it at 4°C and 8000-12000 rpm, and collect the supernatant; (4) Pass the supernatant through an ultrafiltration membrane with a molecular weight cut-off of 3kDa-5kDa, collect the filtrate, and dry it to obtain crude Rana temporaria antifreeze peptide; (5) Prepare a solution of the crude product, load it into a Sephadex G-15 gel chromatography column, use purified water or 0.1mol / L acetic acid solution as eluent, control the flow rate at 0.5-1.0mL / min, collect the main peak components with a relative molecular mass of 500-2000Da in the elution curve, and dry it to obtain purified Rana temporaria antifreeze peptide.

7. Use of the Rana temporaria antifreeze peptide according to any one of claims 1-5 in the preparation of a cosmetic or skin external preparation for preventing or treating skin frostbite and chapping.

8. Use of the Rana temporaria antifreeze peptide according to any one of claims 1-5 in the preparation of a food freezing protective agent, particularly for improving the texture and water holding capacity of meat products or fruits and vegetables during storage.

9. Use of the Rana temporaria-freeze resistant peptide according to any one of claims 1-5 in the preparation of a food or a drug for improving the cold resistance of the body or relieving low-temperature exercise fatigue.

10. An anti-freezing composition, characterized by, comprising: (a) an effective amount of the Rana temporaria-freeze resistant peptide according to any one of claims 1-5; and (b) one or more pharmaceutically or cosmetically acceptable adjuvants selected from at least one of hyaluronic acid, glycerol, propylene glycol, trehalose, and ceramide; the composition is a skin external preparation, a health food, or an anti-freezing spray for the surface of an article; and the mass percentage of the Rana temporaria-freeze resistant peptide in the composition is 0.1% to 20.0%.

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