Preparation method and application of frog oil

Abstract

The application discloses a preparation method and application of frog oil. The frog oil with low acid value, rich in polyunsaturated fatty acids, EPA and DHA and high quality is obtained by adopting enzyme hydrolysis and refining treatment with bullfrog viscera as raw materials, and the quality reaches the national fish oil refining level standard. The obtained frog oil has the unsaturated fatty acid content of more than 70%, the total amount of essential fatty acids, DHA and EPA is increased, the fishy smell is obviously reduced, and the oil aroma quality is obviously improved. The aging mouse model proves that the refined frog oil prepared by the application can enhance the antioxidant enzyme activity of the body, inhibit the generation of malondialdehyde, and inhibit the release of pro-inflammatory factors, so as to improve the antioxidant capacity and anti-inflammatory effect of the body in multiple ways. The frog oil prepared by the application can be used as high-quality raw materials of functional food, functional oil additive and the like.

Description

Technical Field

[0001] This invention relates to the field of high-value utilization of biological resources and preparation of functional oils, specifically to a method for preparing high-quality frog oil using bullfrog visceral by-products as raw materials through enzymatic hydrolysis and synergistic refining. Background Technology

[0002] During the processing of bullfrogs, their internal organs (such as the liver and digestive tract) are often discarded or treated as low-value byproducts, resulting in resource waste. It is worth noting that bullfrog visceral byproducts, especially the liver, are rich in polyunsaturated fatty acids such as DHA and EPA, possessing various physiological activities such as improving brain function, protecting eyesight, and preventing cardiovascular diseases. Therefore, bullfrog viscera are an excellent source for developing functional natural oils. However, the traditional preparation process of frog oil (such as oil extracted from frog bodies) often suffers from problems such as low oil quality, strong off-odors (such as fishy or oxidized smells), and poor oxidative stability, particularly high peroxide values, high saturated fatty acid content, and low retention of unsaturated fatty acids. Currently, there is a lack of a frog oil preparation method that can comprehensively solve the above problems and possess high quality and low off-odor characteristics.

[0003] Therefore, developing a method for preparing frog oil using bullfrog viscera byproducts as raw materials, which has high unsaturated fatty acid content, low peroxide value, and low odor, is of great practical significance for realizing the high-value utilization of bullfrog byproducts and expanding the application scenarios of refined frog oil. Summary of the Invention

[0004] The present invention aims to solve the above-mentioned technical problems and provide a method for preparing high-quality frog oil based on bullfrog visceral by-products, so as to realize the full-value utilization of resources and the functional improvement of oil.

[0005] This invention adopts the following technical solution: a method for preparing frog oil: (1) Enzymatic hydrolysis pretreatment: using bullfrog visceral by-products as raw materials, after crushing and homogenizing, enzymatic hydrolysis was performed using protease to obtain the enzymatic hydrolysis solution; (2) Co-separation treatment: The enzymatic hydrolysis solution is centrifuged and the upper oil phase is collected to obtain crude frog oil; (3) Refining process: The crude frog oil is subjected to phosphoric acid treatment, NaOH treatment, water washing and activated clay treatment in sequence to obtain refined frog oil.

[0006] Furthermore, the enzyme addition amount is 0.5%~2.5%, the enzymatic hydrolysis time is 0.5 h~2.5 h, the enzymatic hydrolysis temperature is 40℃~60℃, the material-liquid ratio (w / v) is 1:1~1:5, and the enzymatic hydrolysis pH is 5.0~9.0.

[0007] Preferably, in step (1), the amount of enzyme added is 1.5%~2.5%, the enzymatic hydrolysis time is 1.5 h~2.5 h, the enzymatic hydrolysis temperature is 45℃~55℃, and the enzymatic hydrolysis pH is 7.0.

[0008] Preferably, in step (1), the protease is papain.

[0009] Furthermore, the centrifugation rate is 8000-10000 rpm, and the centrifugation time is 10-20 min.

[0010] Furthermore, the refining process specifically involves: weighing a certain amount of crude frog oil and heating it to 70°C, adding 1% (w / w) phosphoric acid, heating for 1 min, centrifuging for 10 min to collect the filtrate, heating to 70°C and adding 0.4% NaOH, heating for 30 min, centrifuging for 10 min to recover the supernatant, adding 10% (v / v) deionized water to wash and centrifuging for 5 min, heating to 60°C and adding 15% (w / w) activated clay, reacting for 30 min and centrifuging for 10 min to recover the supernatant.

[0011] The frog oil prepared by the method described above also falls within the scope of protection of this invention.

[0012] The frog oil prepared by the method described herein is used as a functional oil component and can be used to prepare health foods or functional foods with effects such as improving intelligence and brain function, protecting eyesight, delaying aging, improving oxidative stress, and anti-inflammatory effects.

[0013] This invention includes at least the following technical innovations and beneficial effects: (1) Raw material innovation: For the first time, bullfrog viscera by-products are used as raw materials to replace traditional frog body oil extraction, realizing high-value utilization of by-products and reducing waste and environmental pollution.

[0014] (2) Process innovation: The enzymatic hydrolysis method is used in conjunction with refining (including phosphoric acid and NaOH treatment, water washing, and activated clay treatment). The peroxide value of the frog oil is significantly reduced, the fishy smell is significantly reduced, the overall aroma quality is improved, the saturated fatty acid content is reduced to 28.60%, and the unsaturated fatty acid content is increased to 71.40%.

[0015] (3) Functional innovation: The D-galactose-induced aging mouse model confirmed that the frog oil of the present invention can enhance the activity of antioxidant enzymes, inhibit the production of malondialdehyde, and inhibit the release of pro-inflammatory factors, thereby improving the body's antioxidant capacity and anti-inflammatory effect through multiple pathways.

[0016] (4) Beneficial effects: The frog oil obtained by this invention meets the national fish oil refining grade standard, has very low fishy smell, pleasant aroma, and good antioxidant and anti-inflammatory effects. It can provide good raw materials for the development of functional foods, functional pet products and functional cosmetics. Attached Figure Description

[0017] Figure 1 The figure shows the effect of refined frog oil on the antioxidant capacity of aging mice in Example 6.

[0018] Figure 2 The figure shows the effect of refined frog oil on the anti-inflammatory effect of aging mice in Example 6. Detailed Implementation

[0019] To make the above-mentioned objects, features and advantages of the present invention more apparent and understandable, the specific embodiments of the present invention will be further described below with reference to the accompanying drawings.

[0020] Example 1: A crude oil derived from bullfrog viscera byproducts is prepared by the following method: After thawing, washing, and draining, bullfrog viscera were homogenized at 3000 rpm for 3 min at a material-to-liquid ratio of 1:1 (g / mL) to obtain a homogenized viscera sample. Papain was then added at concentrations of 0.5%, 1.0%, 1.5%, 2.0%, and 2.5%. The pH of the solution was adjusted to 7.0, followed by enzymatic hydrolysis at 50℃ for 2 h. After hydrolysis, the enzyme was inactivated by boiling in a water bath for 10 min, followed by centrifugation at 8000 rpm for 15 min. The upper oil phase was collected to obtain crude bullfrog oil.

[0021] Example 2: A crude oil derived from bullfrog viscera byproducts was prepared using a method different from that in Example 1, by fixing the enzyme addition amount at 2.0% and varying the enzymatic hydrolysis time to 0.5, 1.0, 1.5, 2.0, and 2.5 h. The rest of the preparation method remained the same as in Example 1.

[0022] Example 3: A crude oil derived from bullfrog viscera byproducts was prepared using a method different from that in Example 2, except that the enzymatic hydrolysis time was fixed at 2 h, while the hydrolysis temperature was varied to 40, 45, 50, 55, and 60 °C. The rest of the preparation method was the same as in Example 2.

[0023] Example 4: The crude frog oil prepared in Example 3 was refined. A certain amount of crude frog oil was weighed and heated to 70°C. 1% (w / w) of phosphoric acid was added. After heating for 1 min, the mixture was centrifuged for 10 min to collect the filtrate. After heating to 70°C, 0.4% NaOH was added. After heating for 30 min, the mixture was centrifuged for 10 min to recover the supernatant. After washing with 10% (v / v) deionized water, the mixture was centrifuged for 5 min. After heating to 60°C, 15% (w / w) activated clay was added. After reacting for 30 min, the mixture was centrifuged for 10 min to recover the supernatant.

[0024] Example 5: (1) Determination of frog oil extraction rate The total oil content of bullfrog skin and viscera was first determined by the chloroform-methanol method, and the frog oil extraction rate was calculated according to Formula 1: Frog oil extraction rate = m / M × 100% Formula (1) m: Determination of frog oil content under different enzymatic hydrolysis conditions M: Determination of frog oil content by chloroform-methanol method (2) Determination of physicochemical properties of frog oil Acid value (AV) was determined in accordance with GB 5009.229-2016 "National Food Safety Standard - Determination of Acid Value in Food"; peroxide value (PV) was determined in accordance with GB 5009.227-2016 "National Food Safety Standard - Determination of Peroxide Value in Food"; iodine value (IV) was determined in accordance with GB / T 5532-2022 "Determination of Iodine Value in Animal and Vegetable Oils".

[0025] (3) Determination of fatty acid composition of frog oil Add 5 mL of potassium hydroxide-methanol solution (0.5 mol / L) to frog oil, reflux at 100 °C for 10 min, add 3 mL of boron trifluoride-methanol solution (14%), react for 3 min, then add 2 mL of n-hexane, reflux for 2 min, cool to room temperature, add 10 mL of saturated sodium chloride solution, and allow to stand for separation. Collect the upper layer, add an appropriate amount of anhydrous sodium sulfate to dehydrate and dry, filter through a 0.22 μm organic filter membrane, and analyze by GC. A gas chromatography system equipped with a DB-5 capillary column (30 m × 0.32 mm × 0.25 μm) and a flame ionization detector was used. The analytical conditions were set as follows: nitrogen (99.999%) as carrier gas, splitless, injection volume 1 μL, column temperature program: initial temperature 100 °C, hold for 10 min, increase to 180 °C at a rate of 10 °C / min, hold for 5 min, then continue to increase to 200 °C at a rate of 1 °C / min, hold for 20 min. Qualitative identification was performed using the retention time of fatty acid methyl ester standards, and the relative content of each component was calculated using the peak area normalization method.

[0026] (4) Determination of volatile compounds in frog oil Accurately weigh 2.00 g of frog oil into a 20 mL headspace vial, add 2 µL of 0.11 mg / mL methyl nonanoate standard solution as an internal standard, and equilibrate in a 65℃ water bath for 30 min. Simultaneously, insert the extraction head into the headspace vial for headspace adsorption. After extraction, quickly insert the extraction head into the gas chromatograph injection port and thermally desorb at 250℃ for 5 min. Chromatographic analysis was performed on a gas chromatograph equipped with a DB-5 capillary column (30 m × 0.32 mm × 0.25 μm). The column oven was programmed with the following temperature increases: initial temperature 40℃, held for 4 min; increased at 2℃ / min to 90℃, held for 1 min; increased at 8℃ / min to 160℃, held for 1 min; increased at 15℃ / min to 230℃, held for 1 min. Mass spectrometry conditions: Electron ionization (EI) source, ionization energy 70 eV; ion source temperature 230℃, quadrupole temperature 150℃; mass scan range m / z 30–550. The obtained mass spectra were automatically matched with spectra in standard libraries (NIST2014 and Wiley 9) to identify detected volatile compounds. Aroma activity value (OAV) is used to describe the odor activity of volatile compounds. Generally, compounds with an OAV greater than or equal to 1 are considered to contribute significantly to aroma, and the degree of contribution is directly proportional to their OAV.

[0027] Example 6: Evaluation of the biological efficacy of refined frog oil (1) Grouping and dosage design of experimental animals Sixty SPF-grade Kunming mice (4 weeks old, weighing 20.0±2.0 g), half male and half female, were provided by Shanghai Jiesijie Laboratory Animal Co., Ltd. After one week of acclimatization under standard conditions (25℃, 12 h light), all mice were randomly divided into 6 groups of 10 mice each. The dosage for the experimental groups was based on the total DHA and EPA intake recommended in the *Chinese Dietary Reference Intakes (2023 Edition)*. The 6 experimental groups were: blank control group, model group, control group, and refined frog oil intervention groups (low-dose, medium-dose, and high-dose groups). Except for the blank control group, all other groups were injected with D-galactose (125 mg / kg / body weight) solution. All mice were fed standard feed and had free access to water, and were administered gavage at fixed times daily. After 8 weeks of gavage intervention, the mice were fasted for 12 h. Subsequently, blood was collected via the eyes, and the mice were euthanized by cervical dislocation to obtain liver and skin tissue. The collected blood was allowed to stand at 4°C for 2 hours, and then centrifuged at 3500 rpm for 10 minutes at 4°C to obtain serum. The specific dosage is shown in the table below.

[0028] Table 1. Grouping and drug dosage of experimental mice (2) Determination of serum biochemical indicators The levels of superoxide dismutase (SOD), catalase (CAT), malondialdehyde (MDA), total antioxidant capacity (T-AOC), interleukin-1β (IL-Iβ), interleukin-6 (IL-6), interleukin-12 (IL-12), and tumor necrosis factor-α (TNF-α) in serum were determined according to the instructions of the corresponding kits.

[0029] As shown in Table 2, after refining, both the AV and PV of frog oil decreased significantly. P <0.05), while IV increased significantly ( P <0.05). This indicates that the content and quality of unsaturated fatty acids in frog oil were effectively improved. Specifically, the AV and PV of refined frog oil decreased to 1.43 mg / g and 2.43 mmol / kg, respectively, while IV significantly increased from 125.01 g / 100g to 159.56 g / 100g (P<0.05). This suggests that frog oil has a higher content of unsaturated fatty acids and a lower degree of oxidation, resulting in better quality. Notably, the quality of refined frog oil can reach the refined grade specified in national standards.

[0030] Table 2 Effects of refining treatment on the physicochemical properties of frog oil Note: Different letter identifiers represent significant differences (p<0.05). Table 3 shows that after refining, the content of saturated fatty acids (SFA) in frog oil decreased significantly, while the content of monounsaturated fatty acids (MUFA) and polyunsaturated fatty acids (PUFA) increased significantly. This indicates that refining can effectively improve the nutritional value of frog oil. Refining primarily increases the PUFA content in frog oil. Among them, C20:5 (EPA) and C22:6 (DHA) are important functional fatty acids with various biological benefits. Refining effectively promotes the content of C20:5 and C22:6 in frog oil.

[0031] Table 3 Effect of refining treatment on fatty acid composition of frog oil Depend on Figure 1 It can be seen that frog oil has an effect on the antioxidant capacity of aging model mice.

[0032] (1) such as Figure 1As shown in Figure A, the total oxygen saturation (T-AOC) of mice induced by D-galactose was significantly reduced. After gavage administration of different doses of frog oil, their T-AOC gradually returned to normal levels. The high-dose group (20.5 U / mL) and the medium-dose group (17.30 U / mL) both had higher T-AOC levels than the fish oil control group (16.03 U / mL). This indicates that frog oil can effectively restore T-AOC in aging mice, and its effect is superior to that of fish oil.

[0033] (2) such as Figure 1 As shown in Figure B, the SOD content in the model group (6.60 U / mL) was significantly lower than that in the control group (12.48 U / mL). After gavage administration of frog oil, the SOD content in each dose group increased in a dose-dependent manner, reaching 11.36 U / mL in the high-dose group and 10.45±0.31 U / mL in the fish oil control group. This suggests that frog oil can enhance SOD activity and improve free radical scavenging ability.

[0034] (3) such as Figure 1 As shown in Figure C, the CAT activity in the model group (5.55 U / mL) was significantly lower than that in the control group (12.69 U / mL). After administration of frog oil, CAT activity increased in a dose-dependent manner, reaching 10.71 U / mL in the high-dose group, while the medium-dose group (8.83 U / mL) was similar to that in the fish oil control group (8.98 U / mL). The high-dose frog oil group showed the best effect, indicating that it can enhance CAT activity in aging mice.

[0035] (4) such as Figure 1 As shown in Figure D, the MDA content in the model group (6.58 nmol / mL) was significantly higher than that in the blank group, indicating increased lipid peroxidation. After frog oil intervention, the MDA levels in the low, medium, and high dose groups decreased to 5.27, 3.90, and 2.82 nmol / mL, respectively, all significantly lower than those in the model group (…). P The concentration of phospholipids in the frog oil group was less than 0.05%, and even lower in the high-dose group than in the fish oil control group. This indicates that frog oil can effectively inhibit lipid peroxidation and reduce MDA accumulation.

[0036] Frog oil significantly enhanced the antioxidant capacity of aging mice in a dose-dependent manner, manifested by increased T-AOC, SOD, and CAT activities and decreased MDA. The high-dose group showed the best effect and was generally superior to the fish oil control group. This indicates that frog oil has good antioxidant and anti-inflammatory activities.

[0037] Depend on Figure 2 It can be seen that frog oil has an effect on the inflammatory response of aging model mice.

[0038] (1) such as Figure 2 As shown in Figure A, the serum IL-6 level in the blank group mice was 38.89 pg / mL, while in the model group it significantly increased to 114.60 pg / mL after D-galactose induction. P<0.05). After gavage administration of frog oil, the IL-6 levels in the low, medium, and high dose groups decreased to 93.62, 80.43, and 54.05 pg / mL, respectively, all significantly lower than those in the model group, showing a dose-response relationship. The fish oil control group had a level of 75.64 pg / mL, falling between the medium and high dose groups. This indicates that frog oil can effectively reduce IL-6 levels in aging mice and alleviate inflammatory responses.

[0039] (2) such as Figure 2 As shown in B, the IL-12 content in the blank group was 232.98 pg / mL, while that in the model group increased to 375.32 pg / mL. P <0.05). After frog oil intervention, the IL-12 levels in the low, medium, and high dose groups were 328.56, 300.86, and 268.15 pg / mL, respectively, all significantly lower than those in the model group, showing a dose-dependent decrease. The fish oil control group (296.67 pg / mL) showed similar effects to the medium dose group. This suggests that frog oil can inhibit IL-12 overexpression, block excessive activation of the Th1 immune response, and alleviate inflammation.

[0040] (3) such as Figure 2 As shown in C, the TNF-α content in the blank group was 876.924 pg / mL, while that in the model group significantly increased to 1433.58 pg / mL. P <0.05). After frog oil intervention, TNF-α levels in all dose groups were significantly lower than those in the model group, and decreased gradually with increasing dose. The fish oil control group showed a decrease to 1086.92 pg / mL. This indicates that frog oil can effectively inhibit the expression of the core pro-inflammatory factor TNF-α and reduce inflammatory damage.

[0041] (4) such as Figure 2 As shown in Figure D, the IL-1β level in the blank group was 1864.92 pg / mL, while that in the model group increased to 3370.74 pg / mL. P <0.05). After fish oil intervention, the level decreased to 2423.06 pg / mL. The IL-1β levels in all frog oil dosage groups were significantly lower than those in the model group and comparable to those in the blank group. This indicates that frog oil can effectively reduce IL-1β overexpression, inhibit inflammatory initiation signals, and alleviate the inflammatory response.

[0042] Frog oil significantly reduced the overexpression of IL-6, IL-12, TNF-α, and IL-1β in the serum of aging mice in a dose-dependent manner, with an anti-inflammatory effect superior to or equivalent to that of the fish oil control group. This indicates that frog oil possesses good anti-inflammatory activity and the potential to alleviate inflammation-related symptoms.

[0043] Comparative Example 1: Steaming / Boiling Method 20 g of bullfrog viscera were mixed with an equal volume of deionized water and steamed in an 85°C water bath for 1 hour. The supernatant oil was collected by centrifugation at 4000 rpm for 10 minutes to obtain crude bullfrog oil. The crude bullfrog oil was then refined using the same method as in Example 4.

[0044] Comparative Example 2: Organic Solvent Method 20 g of bullfrog viscera were extracted with hexane-isopropanol (3:2, v / v) at a material-to-liquid ratio (g / mL) of 1:5 to 1:20 for 2 h. The supernatant was collected by centrifugation at 5000 rpm for 10 min and concentrated to constant weight in a rotary evaporator at 50 °C to obtain crude frog oil. The crude frog oil was then refined using the same method as in Example 4.

[0045] As shown in Table 4, the organic solvent method exhibited the highest extraction rate (84.13%), followed by the enzymatic hydrolysis method (71.87%), with the lowest being the cooking method (43.48%). Although the organic solvent method had the highest yield, its high risk of organic reagent residue limits its application in functional foods, pet food, and cosmetics. While the yield of the enzymatic hydrolysis method was lower than that of the organic solvent method, it was still as high as 71.87%. The crude frog oil prepared by the enzymatic hydrolysis method had the lowest peroxide value and the highest iodine value, indicating that the frog oil prepared by enzymatic hydrolysis had a lower degree of oxidative deterioration and a higher content of unsaturated fatty acids. Furthermore, after refining, the frog oil prepared by the enzymatic hydrolysis method met the quality requirements for refined fish oil in the national standard for acid value, peroxide value, and iodine value, with the iodine value significantly increasing to 159.56 g / 100g. P The concentration of unsaturated fatty acids (<0.05) indicates a further increase in the content of unsaturated fatty acids. Therefore, enzymatic hydrolysis ensures a high frog oil extraction rate while producing the best quality frog oil.

[0046] Table 4. Effects of different preparation methods on frog oil extraction rate and physicochemical properties Note: Different letters indicate significant differences. p <0.05) As shown in Table 5, although the organic solvent method has the highest frog oil extraction rate, its content of monounsaturated fatty acids (MUFA) and polyunsaturated fatty acids (PUFA) is significantly lower than that of the enzymatic hydrolysis method. More importantly, the crude frog oil prepared by the enzymatic hydrolysis method has higher contents of oleic acid (C18:1) (23.49%), linoleic acid (C18:2) (8.65%), EPA (C20:5) (4.57%), and DHA (C22:6) (9.66%) than that prepared by the organic solvent method. This indicates that the enzymatic hydrolysis method can obtain frog oil with high UFA (especially PUFA) content. In the frog oil prepared by the cooking method, the saturated fatty acid (SFA) content is significantly higher than that prepared by the organic solvent method and the enzymatic hydrolysis method, while the UFA (especially PUFA) content is significantly lower than both, indicating that its fatty acid quality is the worst. After refining, the SFA content of all three types of frog oil decreased significantly, while the MUFA and PUFA contents increased significantly, indicating that refining can effectively improve the nutritional value of frog oil. Among them, the refined frog oil prepared by enzymatic hydrolysis has the highest levels of MUFA (36.71%) and PUFA (34.69%), and the lowest level of SFA (28.60%). Furthermore, its oleic acid, linoleic acid, and DHA contents are all higher than those prepared by organic reagent methods. In summary, frog oil prepared by enzymatic hydrolysis has the best fatty acid quality and can be used as a high-quality raw material for functional oils.

[0047] Table 5. Effects of different preparation methods on the fatty acid composition of frog oil Note: Different letter identifiers represent significant differences (p<0.05). As shown in Table 6, (E, E)-2,4-heptadenal is the key compound constituting the fishy odor of frog oil. Among the three types of crude frog oil, (E, E)-2,4-heptadenal exhibited high aroma activity values ​​(OAV), indicating a strong fishy odor. Notably, the crude fish oil prepared by the cooking method exhibited the strongest fishy odor, while the crude fish oil prepared by the organic reagent method and the enzymatic hydrolysis method showed the lowest fishy odor and the highest overall aroma, respectively. After refining, the OAV of (E, E)-2,4-heptadenal in both the organic reagent method and the enzymatic hydrolysis method significantly decreased, indicating a significant reduction in fishy odor. Furthermore, in the enzymatically refined fish oil, the OAV of E-2-octenal and nonanal, which possess typical oil flavors, was significantly higher than that in the organically refined fish oil. Therefore, frog oil prepared by the enzymatic hydrolysis method not only has a lower fishy odor but also possesses the best aroma quality, laying a good raw material foundation for the diversified development of frog oil.

[0048] Table 6. Effects of different preparation methods on the aroma activity values ​​of frog oil flavor compounds

Claims

1. A method for preparing frog oil, characterized in that, Includes the following steps: (1) Enzymatic hydrolysis pretreatment: using bullfrog visceral by-products as raw materials, after crushing and homogenizing, enzymatic hydrolysis was performed using protease to obtain the enzymatic hydrolysis solution; (2) Co-separation treatment: The enzymatic hydrolysis solution is centrifuged and the upper oil phase is collected to obtain crude frog oil; (3) Refining process: The crude frog oil is subjected to phosphoric acid treatment, NaOH treatment, water washing and activated clay treatment in sequence to obtain refined frog oil.

2. The preparation method according to claim 1, characterized in that, In step (1), the enzyme addition amount is 0.5%~2.5%, the enzymatic hydrolysis time is 0.5 h~2.5 h, the enzymatic hydrolysis temperature is 40℃~60℃, the material-liquid ratio (w / v) is 1:1~1:5, and the enzymatic hydrolysis pH is 5.0~9.

0.

3. The preparation method according to claim 2, characterized in that, In step (1), the amount of enzyme added is 1.5%~2.5%, the enzymatic hydrolysis time is 1.5 h~2.5 h, the enzymatic hydrolysis temperature is 45℃~55℃, and the enzymatic hydrolysis pH is 7.

0.

4. The preparation method according to claim 1, characterized in that, In step (1), the protease is papain.

5. The preparation method according to claim 1, characterized in that, In step (2), the centrifugation rate is 8000-10000 rpm and the centrifugation time is 10-20 min.

6. The preparation method according to claim 1, characterized in that, In step (3), the refining process is as follows: a certain amount of crude frog oil is weighed and heated to 70°C, 1% (w / w) of phosphoric acid is added, heated for 1 min and centrifuged for 10 min to collect the filtrate, heated to 70°C and 0.4% of NaOH is added, heated for 30 min and centrifuged for 10 min to recover the upper layer solution, 10% (v / v) of deionized water is added for washing and centrifuged for 5 min, heated to 60°C and 15% (w / w) of activated clay is added, reacted for 30 min and centrifuged for 10 min to recover the upper layer solution.

7. Frog oil prepared by the method according to any one of claims 1 to 6.

8. The use of the frog oil according to claim 7, characterized in that, The frog oil, as a functional oil component, can be used to prepare health foods or functional foods with effects such as improving intelligence and brain function, protecting eyesight, delaying aging, improving oxidative stress, and anti-inflammation.

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