An enzymatic extraction process for oil from the spiny-breasted frog.

Abstract

This invention relates to the field of biology, specifically to an enzymatic extraction process for spiny-breasted frog oil. The extraction process includes the following steps: Step S1: raw material pretreatment; Step S2: ultrasound-assisted compound enzymatic hydrolysis; Step S3: biphasic extraction and primary separation. This invention systematically integrates and innovates multiple technologies, including directional raw material pretreatment, ultrasound-assisted biomimetic enzymatic hydrolysis, green biphasic extraction, and low-temperature molecular distillation purification, to construct a complete and efficient combined production line. This improves oil extraction efficiency and product purity, maximizes the retention of heat-sensitive active ingredients, and enhances the added value of the product through functional design. Simultaneously, it achieves near-full elemental resource utilization of raw materials, forming a green and high-value production model with broad application prospects in high-end health foods, cosmetics, pharmaceutical excipients, and nutritional supplements.

Description

Technical Field

[0001] This invention relates to the field of biology, and more particularly to an enzymatic extraction process for oil from the spiny-breasted frog. Background Technology

[0002] Spiny-breasted frog oil is a type of natural animal fat extracted from the adipose tissue of the spiny-breasted frog. It is rich in polyunsaturated fatty acids, phospholipids, and fat-soluble active substances. In traditional medicine, this oil is believed to have the effects of nourishing yin and moistening the lungs, promoting tissue regeneration and wound healing. In the modern health industry, as a high-value-added raw material, it shows broad application prospects in the fields of high-end functional foods, skin care products, and biopharmaceutical preparations. However, its extraction technology still has many limitations, which restrict the quality and overall benefits of the product.

[0003] Traditional extraction processes often employ organic solvent extraction or high-temperature melting. While organic solvent extraction yields a higher oil yield, it carries the risk of solvent residue and may damage heat-sensitive nutrients in the oil, affecting product safety and bioactivity. High-temperature melting, on the other hand, easily leads to oxidative rancidity of the oil, resulting in low quality.

[0004] To overcome the shortcomings of traditional extraction processes, existing technologies have introduced aqueous enzymatic methods with biological enzymatic hydrolysis as the core. However, the single enzymatic hydrolysis process has limited efficiency in decomposing the dense connective tissue of frogs, has a long extraction cycle, and the stable emulsion system formed after enzymatic hydrolysis is difficult to break and separate, making it difficult to achieve both oil yield and purity. At the same time, existing technologies mostly focus on obtaining single oil products, while neglecting the recovery and utilization of by-products such as protein peptides and minerals rich in enzymatic hydrolysis residues. Therefore, they fail to achieve the high-value conversion of all components of the raw materials. In addition, the crude oil after preliminary extraction usually requires multiple refining steps to meet the standards. The refining process is lengthy and may be accompanied by the loss of active ingredients.

[0005] Therefore, based on the relevant technologies mentioned above, there is an urgent need to develop an enzymatic extraction process for spiny-breasted frog oil. Summary of the Invention

[0006] In view of this, the purpose of this invention is to propose an enzymatic extraction process for spiny-breasted frog oil to solve the problems of low extraction rate, poor quality, loss of activity, and insufficient utilization in the existing technology.

[0007] To achieve the above objectives, the present invention provides an enzymatic extraction process for oil from the spiny-breasted frog.

[0008] An enzymatic extraction process for processing oil from the spiny-breasted frog, comprising the following steps: Step S1: Raw material pretreatment: After cleaning and chopping the adipose tissue of the spiny-breasted frog, it was subjected to directional maturation and freeze-thaw cycle treatment to obtain a pretreated slurry. Step S2: Ultrasonic-assisted compound enzymatic hydrolysis: The slurry obtained in step S1 is mixed with deionized water, and after adjusting the pH and temperature, a compound enzyme is added, and the enzymatic hydrolysis reaction is carried out under the synergistic effect of pulsed ultrasonic field. Step S3: Two-phase extraction and primary separation: The enzyme hydrolysate was inactivated and centrifuged to separate the oil and emulsion mixture. The resulting oil-emulsion mixture was subjected to ethanol-ammonium sulfate two-phase extraction to separate the alcohol phase and recover the ethanol, thus obtaining refined spiny-breasted frog hair oil. At the same time, the mixture of aqueous phase and solid residue was collected. Step S4: Fine separation by molecular distillation: The refined crude oil obtained in step S3 was subjected to short-path molecular distillation to separate the high-purity spiny-breasted frog oil main fraction. Step S5: In-situ reconstruction of functional greases: Lecithin and phytosterols were added to the main fraction of high-purity spiny-breasted frog oil obtained in step S4, and homogenization was carried out to obtain self-emulsifying functional spiny-breasted frog oil. Step S6: Full utilization of by-products: The aqueous phase and solid residue mixture obtained in step S3 were extracted, concentrated and dried to obtain spiny-breasted frog active peptide powder and calcium-phosphorus complex.

[0009] Pre-treatment of the slurry creates favorable conditions for efficient enzymatic hydrolysis. Meanwhile, the hydrolysis products are separated in a green manner to obtain pure oils, which are then physically refined and functionalized to obtain stable and high-value end products. In addition, the waste in traditional processes is transformed into economically valuable by-products, which greatly improves resource utilization and overall economic benefits, and embodies the concepts of circular economy and green manufacturing.

[0010] Preferably, the directional curing in step S1 specifically involves cooling the slurry to 0-4°C, maintaining a humidity of 80-90%, and allowing it to stand for 16-20 hours. The freeze-thaw cycle treatment in step S2 specifically involves cooling the directional matured slurry to -35 to -45°C for 100 to 140 minutes, then heating it to 0 to 4°C for 50 to 70 minutes, and repeating the above operation 2 to 4 times.

[0011] By using low temperature and high humidity conditions and the natural enzyme system of adipose tissue for mild biotransformation, some tissues can be initially decomposed and flavor precursors can be released. This process also softens cell structures, making subsequent enzymatic hydrolysis easier. At the same time, the volume expansion effect of water forming ice crystals physically punctures cell membranes and organelle membranes, making it easier to release intracellular lipids and contents, further improving mass transfer efficiency and reducing enzyme dosage and reaction time. In addition, the combination of these two methods creates an excellent substrate state for subsequent enzymatic hydrolysis reactions, which is a fundamental prerequisite for ensuring the high efficiency of the entire process.

[0012] Preferably, the mass ratio of the slurry to deionized water in step S2 is 0.4-0.6:1.

[0013] Preferably, the complex enzyme agent in step S2 includes a 1,3-position specific lipase, a neutral protease, and a chitinase.

[0014] Preferably, based on the mass of the slurry, the amount of the compound enzyme system added is: 0.4%-0.6% 1,3-position specific lipase, 0.8%-1.2% neutral protease, and 0.08%-0.12% chitinase.

[0015] Preferably, in the enzymatic hydrolysis reaction described in step S2, the reaction temperature is 45-55℃ and the reaction time is 160-200 min; The pH adjustment in step S2 specifically involves adding 0.5 mol / L citrate-sodium citrate buffer solution to adjust the pH to 6.4-6.6.

[0016] By setting a near-neutral pH and a moderate temperature, the optimal activity ranges of lipase and protease can be simultaneously considered, while maximizing the protection of heat-sensitive and acid-sensitive active ingredients in oils. Furthermore, the microjets and shock waves generated by ultrasonic cavitation greatly enhance liquid-solid mass transfer, making it easier for enzyme molecules to bind to substrates. The pulse mode effectively controls the temperature rise of the reaction system, avoiding local overheating that could lead to enzyme inactivation or oil oxidation. In addition, through the synergistic effect of both, the rate and uniformity of the enzymatic hydrolysis reaction are significantly improved, shortening the reaction time from several hours to a shorter time. The overall conditions are mild, perfectly balancing efficiency and quality.

[0017] Preferably, the parameters of the pulsed ultrasonic field in step S2 are: frequency 35-45Hz, power density 45-55W / L, and working mode of ultrasonic 4-6s with interval 8-12s.

[0018] Preferably, the ethanol-ammonium sulfate biphase extraction in step S3 specifically involves: adding an 85% aqueous ethanol solution to the oil-emulsion mixture, heating to 35-45°C, stirring at 100-200 rpm for 15-25 minutes, adding ammonium sulfate, allowing it to stand for 40-60 minutes, and then taking the alcohol phase after separation. The mass ratio of the oil-emulsion mixture, the ethanol-water solution, and the ammonium sulfate is 1:1.4-1.6:0.37-0.38.

[0019] By using an ethanol-water solution, oils can be effectively dissolved. At the same time, it has a certain degree of miscibility with the aqueous phase, which facilitates the formation of a two-phase system. Furthermore, by adding ammonium sulfate, a "salting-out effect" is generated, which reduces the solubility of ethanol in water and promotes rapid and clear phase separation of the system. This allows for the efficient separation of oils (dissolved in the alcohol phase) from most water-soluble impurities such as proteins and polysaccharides (remaining in the salt phase).

[0020] Preferably, the short-range molecular distillation in step S4 is a two-stage distillation. The first stage conditions are: evaporation temperature 110-130℃, vacuum degree 0.8-1.0 Pa, and the second stage conditions are: evaporation temperature 170-190℃, vacuum degree 0.3-0.5 Pa.

[0021] By using a vacuum system, the boiling point of the material can be significantly reduced, allowing the polyunsaturated fatty acids in the spiny-breasted frog oil to be separated at a temperature far below its atmospheric decomposition temperature. The two-stage distillation process further improves the flavor and stability of the oil, while also yielding extremely high-purity spiny-breasted frog oil. The combination of these two processes further enhances the quality of the final product.

[0022] Preferably, the amount of lecithin added in step S5 is 4%-6% of the mass of the main fraction of high-purity spiny-breasted frog oil, and the amount of phytosterol added is 1.5%-2.5%. The homogenization process described in step S5 is performed under the following conditions: nitrogen atmosphere, temperature 55-65℃, rotation speed 8000-12000 rpm, for 4-6 minutes.

[0023] The beneficial effects of this invention are: This invention provides an enzymatic extraction process for spiny-breasted frog oil. This invention systematically integrates and innovates multiple technologies, including directional pretreatment of raw materials, ultrasonic-assisted biomimetic enzymatic hydrolysis, green biphasic extraction, low-temperature molecular distillation purification, in-situ reconstruction of functional oils, and stepwise extraction of by-products. This constructs a complete and efficient combined production line. Compared with existing technologies, this process significantly improves oil extraction efficiency and product purity, maximizes the preservation of heat-sensitive active ingredients, and enhances the added value of the product through functional design. Simultaneously, it achieves near-full elemental resource utilization of raw materials, forming a green and high-value production model. The high-quality functional oils, active peptides, and mineral products obtained by this process have broad application prospects in high-end health foods, cosmetics, pharmaceutical excipients, and nutritional supplements. Attached Figure Description

[0024] To more clearly illustrate the technical solutions in this invention or the prior art, the drawings used in the description of the embodiments or the prior art will be briefly introduced below. Obviously, the drawings described below are only for this invention. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0025] Figure 1 This is a bar chart showing the determination of oil yield and oil quality in this invention. Figure 2 This is a bar chart showing the emulsification performance measurement in this invention; Figure 3 This invention relates to the determination of peptide content and solubility of active peptide powder. bar chart Figure 4 This is a bar chart showing the determination of calcium and phosphorus content in the calcium-phosphorus complex of the present invention. Detailed Implementation

[0026] To make the objectives, technical solutions, and advantages of this invention clearer, the invention will be further described in detail below with reference to specific embodiments.

[0027] Example 1: An enzymatic extraction process for oil from the spiny-breasted frog. S1: Wash 1000g of spiny-breasted frog fat tissue and chop it into pieces about 3mm thick. 3 The slurry was cooled to 0°C and 90% humidity, left to stand for 16 hours, then cooled to -35°C and treated for 100 minutes, then heated to 4°C and treated for 50 minutes. The above operation was repeated 4 times to obtain the pretreated slurry. S2: Add 400g of pretreated slurry to 1000g of deionized water, mix well, add 0.5mol / L citrate-sodium citrate buffer, adjust the pH to 6.4-6.6, heat to 45℃, add 1.6g of 1,3-position specific lipase, 3.2g of neutral protease and 0.32g of chitinase, place in a pulsed ultrasonic field, frequency 35Hz, power density 55W / L, working mode is ultrasonic 4s, intermittent 12s, reaction 160min, enzymatic hydrolysis is completed, heat to 80℃, continue for 12min, speed 4500rpm, centrifuge for 20min, separate the upper oil-emulsion mixture, lower aqueous phase and solid residue; S3: Add 140g of 85% ethanol aqueous solution to 100g of oil-emulsion mixture, heat to 35℃, stir at 200rpm for 15min, add 37g of ammonium sulfate, stir to dissolve, let stand for 60min, separate the upper alcohol phase, and rotary evaporate to obtain refined spiny-breasted frog hair oil, while collecting the mixture of aqueous phase and solid residue. S4: The refined spiny-breasted frog hairs were subjected to short-path molecular distillation. The first stage of distillation was carried out at 110°C and a vacuum of 1.0 Pa to obtain a light component. The second stage of distillation was carried out at 170°C and a vacuum of 0.5 Pa to obtain the main fraction of high-purity spiny-breasted frog oil. S5: Under a nitrogen atmosphere, 2g of lecithin and 0.75g of phytosterol were added to 50g of high-purity spiny frog oil main fraction, heated to 55℃, rotated at 12000rpm, and treated for 4min to obtain spiny frog oil. S6: Filter the mixture of aqueous phase and solid residue, treat the filtrate with an ultrafiltration membrane system with a molecular weight cutoff of 5 kDa, add maltodextrin, dry to obtain spiny-breasted frog active peptide powder, add the filtered solid residue to 0.1 mol / L sodium hydroxide solution, heat to 50℃, extract for 2 h, centrifuge, wash and dry, pulverize through a 200-mesh sieve to obtain calcium-phosphorus complex powder.

[0028] Example 2: An enzymatic extraction process for oil from the spiny-breasted frog. S1: Wash 1000g of spiny-breasted frog fat tissue and chop it into pieces about 3mm thick. 3 The slurry was cooled to 2°C and 85% humidity, left to stand for 18 hours, then cooled to -40°C and treated for 120 minutes. The temperature was then raised to 2°C and treated for 60 minutes. The above operation was repeated 3 times to obtain the pretreated slurry. S2: Add 500g of pretreated slurry to 1000g of deionized water, mix well, add 0.5mol / L citrate-sodium citrate buffer, adjust the pH to 6.4-6.6, heat to 50℃, add 2.5g of 1,3-position specific lipase, 5g of neutral protease and 0.5g of chitinase, place in a pulsed ultrasonic field, frequency 40Hz, power density 50W / L, working mode is ultrasonic 5s, intermittent 10s, reaction for 180min, enzymatic hydrolysis is completed, heat to 85℃, continue for 10min, speed 5000rpm, centrifuge for 15min, separate the upper oil-emulsion mixture, lower aqueous phase and solid residue; S3: Add 150g of 85% ethanol aqueous solution to 100g of oil-emulsion mixture, heat to 40℃, stir at 150rpm for 20min, add 37.5g of ammonium sulfate, stir to dissolve, let stand for 50min, separate the upper alcohol phase, and rotary evaporate to obtain refined spiny-breasted frog hair oil, while collecting the mixture of aqueous phase and solid residue. S4: The refined spiny-breasted frog hairs were subjected to short-path molecular distillation. The first stage of distillation was carried out at 120°C and a vacuum of 0.9 Pa to obtain a light component. The second stage of distillation was carried out at 180°C and a vacuum of 0.4 Pa to obtain the main fraction of high-purity spiny-breasted frog oil. S5: Under a nitrogen atmosphere, 2.5g of lecithin and 1g of phytosterol were added to 50g of high-purity spiny frog oil main fraction, heated to 60℃, rotated at 10000rpm, and treated for 5min to obtain spiny frog oil. S6: Filter the mixture of aqueous phase and solid residue, treat the filtrate with an ultrafiltration membrane system with a molecular weight cutoff of 5 kDa, add maltodextrin, dry to obtain spiny-breasted frog active peptide powder, add the filtered solid residue to 0.1 mol / L sodium hydroxide solution, heat to 50℃, extract for 2 h, centrifuge, wash and dry, pulverize through a 200-mesh sieve to obtain calcium-phosphorus complex powder.

[0029] Example 3: An enzymatic extraction process for oil from the spiny-breasted frog. S1: Wash 1000g of spiny-breasted frog fat tissue and chop it into pieces about 3mm thick. 3 The slurry was cooled to 4°C and 80% humidity, left to stand for 20 hours, then cooled to -45°C and treated for 140 minutes, then heated to 0°C and treated for 70 minutes. The above operation was repeated twice to obtain the pretreated slurry. S2: Add 600g of pretreated slurry to 1000g of deionized water, mix well, add 0.5mol / L citrate-sodium citrate buffer, adjust the pH to 6.4-6.6, heat to 55℃, add 3.6g of 1,3-position specific lipase, 7.2g of neutral protease and 0.72g of chitinase, place in a pulsed ultrasonic field, frequency 45Hz, power density 45W / L, working mode is ultrasonic 6s, intermittent 8s, reaction for 200min, enzymatic hydrolysis is completed, heat to 90℃, continue for 8min, speed 5500rpm, centrifuge for 10min, separate the upper oil-emulsion mixture, lower aqueous phase and solid residue; S3: Add 160g of 85% ethanol aqueous solution to 100g of oil-emulsion mixture, heat to 45℃, stir at 100rpm for 25min, add 38g of ammonium sulfate, stir to dissolve, let stand for 40min, separate the upper alcohol phase, and rotary evaporate to obtain refined spiny-breasted frog hair oil, while collecting the mixture of aqueous phase and solid residue. S4: The refined spiny-breasted frog hairs were subjected to short-path molecular distillation. The first stage of distillation was carried out at 130°C and a vacuum of 0.8 Pa to obtain a light component. The second stage of distillation was carried out at 190°C and a vacuum of 0.3 Pa to obtain the main fraction of high-purity spiny-breasted frog oil. S5: Under a nitrogen atmosphere, 3g of lecithin and 1.25g of phytosterols were added to 50g of high-purity spiny frog oil main fraction, heated to 65℃, rotated at 8000rpm, and treated for 6min to obtain spiny frog oil. S6: Filter the mixture of aqueous phase and solid residue, treat the filtrate with an ultrafiltration membrane system with a molecular weight cutoff of 5 kDa, add maltodextrin, dry to obtain spiny-breasted frog active peptide powder, add the filtered solid residue to 0.1 mol / L sodium hydroxide solution, heat to 50℃, extract for 2 h, centrifuge, wash and dry, pulverize through a 200-mesh sieve to obtain calcium-phosphorus complex powder.

[0030] Example 4: An enzymatic extraction process for oil from the spiny-breasted frog. Compared with Example 1, this embodiment adds 0.025g of mixed tocopherols and 0.015g of rosemary extract in step S5. All other steps and parameters are the same, and will not be repeated in this embodiment.

[0031] Comparative Example 1: Compared with Example 1, this comparative example did not activate ultrasound assistance in step S2, but the remaining steps and parameters were the same, and will not be repeated here.

[0032] Comparative Example 2: Compared with Example 1, this comparative example did not perform freeze-thaw cycle treatment in step S1. All other steps and parameters are the same, and will not be repeated here.

[0033] Comparative Example 3: This comparative example differs from Example 1 only in that the "complex enzyme" is replaced with an equal amount of "1,3-position specific lipase". All other steps and parameters are the same, and will not be repeated here.

[0034] Comparative Example 4: Compared with Example 1, this comparative example did not use ethanol-ammonium sulfate two-phase extraction in step S3. Instead, the oil-emulsion mixture was directly added to 3 times the volume of 95% ethanol, allowed to stand and precipitate, and the supernatant was taken. The ethanol was recovered to obtain oil. The remaining steps and parameters were the same, and this comparative example will not be repeated.

[0035] Performance testing: Oil yield: (reference) Figure 1 ) Calculation formula:

[0036] Oil quality determination: Acid value: Refer to the testing standard GB 5009.229-2016; 1. Take 5.0 g of oil samples from Examples 1-4 and Comparative Examples 1-4 respectively, add 50 mL of neutralized ether-ethanol mixture, shake well, add 2-3 drops of phenolphthalein indicator, and immediately titrate with 0.1 mol / L potassium hydroxide standard solution until the solution turns slightly red and does not fade within 15 seconds, which is the titration endpoint. At the same time, perform a blank test, that is, without adding oil sample, only titrate with 50 mL of ether-ethanol mixture; 2. Formula for calculating acid value (AV):

[0037] V: Volume of potassium hydroxide standard solution consumed by the sample, in milliliters (mL). V0: Volume of potassium hydroxide standard solution consumed in the blank test, in milliliters (mL); C: The accurate concentration of the potassium hydroxide standard solution, in moles per liter (mol / L). 56.1: Molar mass of potassium hydroxide, in grams per mole (g / mol). m: Sample mass, in grams (g).

[0038] Peroxide value determination: Refer to the testing standard GB 5009.227-2016; 1. Take 2.0 g of oil samples from Examples 1-4 and Comparative Examples 1-4 respectively, add 20 mL of acetic acid-isooctane mixture, shake well, add 1.00 mL of potassium iodide saturated solution, let stand for 5 min, add 75 mL of deionized water, and immediately titrate with 0.1 mol / L sodium thiosulfate standard solution until the solution is pale yellow. Add 0.5 mL of starch indicator, and the solution turns blue. Continue titrating until the blue color just disappears, which is the endpoint. At the same time, perform a blank test, that is, without adding oil sample, only titrate with 20 mL of acetic acid-isooctane mixture; 2. Peroxide value (POV) calculation formula:

[0039] V: Volume of sodium thiosulfate standard solution consumed by the sample, in milliliters (mL). V0: Volume of sodium thiosulfate standard solution consumed in the blank test, in milliliters (mL). C: The accurate concentration of sodium thiosulfate standard, in moles per liter (mol / L). 1000: Hydrogen unit conversion factor; m: Sample mass, in grams (g).

[0040]

[0041] Emulsification performance test: (Reference) Figure 2 ) 1. Determination of Emulsifying Activity Index (EAI) and Emulsifying Stability Index (ESI): 2. Take 1.0 g of each of the oil samples from Examples 1-4 and Comparative Examples 1-4, add 20 mL of 0.1% sodium dodecyl sulfate solution, cool to 25°C, place in a high-speed homogenizer, homogenize at 10000 rpm for 60 s, after homogenization, take a sample, and measure its absorbance at 500 nm using a UV-Vis spectrophotometer, denoted as A0. Let the remaining solution stand at room temperature for 10 min, and measure its absorbance at 500 nm, denoted as A. 10 The blank group was prepared by adding only 0.1% sodium dodecyl sulfate solution. 3. Calculation of Emulsifying Activity Index (EAI):

[0042] .

[0043] .

[0044] .

[0045] .

[0046] .

[0047]

[0048] By-product quality index testing Determination of peptide content in active peptide powder (reference) Figure 3 ) The FOSS Kjeltec 8400 nitrogen analyzer was used. 1. Take 0.5g of each of the active peptide powders from Examples 1-4 and Comparative Examples 1-4, add 5.0g of catalyst mixture (potassium sulfate and copper sulfate, mass ratio 10:1) and 10mL of concentrated sulfuric acid, place on a digestion furnace, first heat at low temperature until foaming stops, then increase the temperature and maintain a gentle boil until the solution turns a transparent blue-green color, continue for 3 hours, cool, add 20mL of ammonia-free water, and obtain the sample; 2. Add 2-3 drops of mixed indicator (methyl red-bromocresol green indicator solution) to 25 mL of boric acid absorption solution. The solution turns purple-red. Take 10.0 mL (recorded as V3) of sample and add it to the reaction chamber of the distillation apparatus. Add 25 mL of 400 g / L sodium hydroxide solution and pass steam through for distillation. When the volume of the distillate is about 150 mL, titrate the absorption liquid in the receiving flask with 0.1 mol / L hydrochloric acid standard solution. The endpoint is when the color changes from blue-green to gray-purple. Record the volume of hydrochloric acid consumed, V1. At the same time, prepare a blank test. Repeat all the above steps except without adding a sample and record the volume of hydrochloric acid consumed, V2.

[0049] 3. Calculation:

[0050] 0.0140: millimolecular mass of nitrogen, g / mmol; m: Sample mass, g; M: Moisture content of the sample (%).

[0051] Determination of the solubility of active peptide powder 1. Take 1.0 g of each of the active peptide powders from Examples 1-4 and Comparative Examples 1-4, add 80 mL of deionized water, heat to 25°C, stir at 200 rpm for 30 min, and bring the volume to 100 mL to obtain the sample solution. 2. Take 50 mL of sample solution, centrifuge at 4000 rpm for 10 min, take 10 mL of supernatant, heat to 105 ℃, dry to constant weight, and record as M1; 3. Calculation:

[0052] M0: Mass of the weighing dish, g; M1: Mass of the weighing dish and the dried solids, in g; m: Sample mass, g.

[0053]

[0054] Determination of calcium and phosphorus content in calcium-phosphorus complex (reference) Figure 4 ) 1. An inductively coupled plasma atomic emission spectrometer is used; Take the calcium-phosphorus complexes from Examples 1-4 and Comparative Examples 1-4 respectively, grind them through a 100-mesh sieve, take 0.1g, add 6mL of nitric acid and 2mL of hydrogen peroxide, let stand for 30min, heat to 120℃, react for 5min, then heat to 180℃, react for 15min, after the reaction is complete, cool, add the digestion solution to a 50mL volumetric flask, and dilute to the mark to obtain the test solution, and at the same time prepare blank reagents; 2. Calcium Standard Series: Prepare standard solutions with concentrations of 0, 0.5, 1.0, 2.0, and 5.0 mg / L by stepwise dilution of the calcium standard stock solution with nitric acid solution (5%). Phosphorus standard series: Prepare standard solutions with concentrations of 0, 0.5, 1.0, 2.0 and 5.0 mg / L by progressively diluting the phosphorus standard stock solution with nitric acid solution (5%).

[0055] 3. Using an inductively coupled plasma atomic emission spectrometer, the blank solution, standard series solutions, and sample solution were measured sequentially. 4. Calculate the concentrations C of calcium and phosphorus:

[0056] C: Concentration of calcium or phosphorus in the sample solution, mg / L; C0: Concentration of calcium or phosphorus in the blank solution, mg / L; V: Sample final volume (50 mL); m: Sample mass, g; 1000: Unit conversion factor.

[0057]

[0058] Data Analysis: As can be seen from Tables 1-4, the spiny-breasted frog oil and related by-products prepared by the process of the present invention have higher oil yield, better oxidative stability and emulsification performance, and the content of active peptides, solubility and mineral recovery rate of by-products are also significantly improved. In contrast, Comparative Example 1, due to the lack of ultrasonic assistance, resulted in insufficient enzymatic hydrolysis, reduced mass transfer efficiency, decreased oil yield, and increased acid value and peroxide value. This was because the enzymatic hydrolysis process lacked the micro-jet and shock wave effects brought about by the cavitation effect, which limited the contact area between the enzyme and the substrate and significantly reduced the mass transfer efficiency. At the same time, the uneven temperature distribution of the reaction system and the local overheating phenomenon aggravated the thermal inactivation of the enzyme and the oxidative rancidity of the oil, resulting in a significant increase in the acid value and peroxide value of the product. In addition, the incomplete enzymatic hydrolysis also affected the dissolution efficiency of active peptides and minerals in the subsequent by-products, resulting in an overall decrease in resource extraction rate. Comparative Example 2, which did not undergo freeze-thaw cycling, resulted in incomplete cell disruption, insufficient lipid release, lower yield, and reduced recovery rates of peptides and minerals from byproducts. This was because the absence of the ice crystal expansion effect limited the release of intracellular lipids, proteins, and minerals, affecting not only the main product yield but also significantly reducing the recovery rates of peptides, calcium, and phosphorus from byproducts. Furthermore, poor substrate accessibility led to prolonged subsequent enzymatic hydrolysis reaction times and increased enzyme requirements, impacting the overall economic efficiency and effectiveness of the process. This demonstrates the fundamental supporting role of freeze-thaw treatment in the pretreatment stage for the overall efficiency of the entire process. Comparative Example 3, due to the use of only a single lipase instead of a complex enzyme system, resulted in insufficient breakdown of protein and chitin structures, significantly reducing the oil extraction rate, peptide content, and calcium and phosphorus recovery rate. This was because the absence of neutral protease and chitinase led to incomplete degradation of the tissue matrix, which not only limited oil dissolution but also affected the dissolution efficiency of peptides and minerals in the aqueous phase, resulting in a significant decrease in the yield and purity of by-products. In addition, the single enzyme system had a limited mechanism of action, lost the synergistic effect of the reaction system, and reduced the overall efficiency and product diversity of the extraction process. This demonstrates the irreplaceable role of the complex enzyme system in achieving the degradation of all tissue components and the comprehensive utilization of resources. Comparative Example 4, due to the absence of ethanol-ammonium sulfate two-phase extraction and the use of a single ethanol precipitation method, resulted in a higher residual amount of water-soluble impurities in the oil, a higher acid value, and decreased emulsion stability. This was attributed to the lack of the salting-out effect of ammonium sulfate, leading to slow and incomplete phase separation, which affected the oil recovery rate. Furthermore, the residual impurities further impacted the effectiveness of subsequent molecular distillation and functionalization modifications. In addition, the presence of impurities also reduced the emulsification properties and storage stability of the oil, highlighting the crucial role of two-phase extraction in ensuring the initial purity and subsequent processing suitability of the oil. Example 4 introduced a mixture of tocopherols and rosemary extract into the in-situ reconstruction step of the functional oil, which significantly enhanced the product's functionality. This addition directly constructed a complex antioxidant system. Tocopherols acted as the main free radical scavenger, while rosemary polyphenols played a synergistic enhancing role. As a result, the oxidative stability of the oil was significantly better than that of other examples, effectively extending the shelf life. At the same time, the reduction of oxidation products maintained the integrity of the oil interface, resulting in the highest emulsion stability index, highlighting the positive impact of antioxidant treatment on application performance. In addition, although the antioxidants did not directly act on the by-product extraction process, the stability of the oil phase reduced the interference of oxidative stress on the extraction process, thereby indirectly leading to a slight increase in the solubility of active peptides and the recovery rate of calcium and phosphorus.

[0059] Those skilled in the art should understand that the discussion of any of the above embodiments is merely exemplary and is not intended to imply that the scope of the invention is limited to these examples; within the framework of the invention, the technical features of the above embodiments or different embodiments can also be combined, the steps can be implemented in any order, and there are many other variations of the different aspects of the invention as described above, which are not provided in detail for the sake of brevity.

[0060] This invention is intended to cover all such substitutions, modifications, and variations that fall within the broad scope of the appended claims. Therefore, any omissions, modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of this invention should be included within the scope of protection of this invention.

Claims

1. An enzymatic extraction process for processing oil from the spiny-breasted frog, characterized in that, The extraction process steps are as follows: Step S1: Raw material pretreatment: After cleaning and chopping the adipose tissue of the spiny-breasted frog, it was subjected to directional maturation and freeze-thaw cycle treatment to obtain a pretreated slurry. Step S2: Ultrasonic-assisted compound enzymatic hydrolysis: The slurry obtained in step S1 is mixed with deionized water, and after adjusting the pH and temperature, a compound enzyme is added, and the enzymatic hydrolysis reaction is carried out under the synergistic effect of pulsed ultrasonic field. Step S3: Two-phase extraction and primary separation: The enzyme hydrolysate was inactivated and centrifuged to separate the oil and emulsion mixture. The resulting oil-emulsion mixture was subjected to ethanol-ammonium sulfate two-phase extraction to separate the alcohol phase and recover the ethanol, thus obtaining refined spiny-breasted frog hair oil. At the same time, the mixture of aqueous phase and solid residue was collected. Step S4: Fine separation by molecular distillation: The refined crude oil obtained in step S3 was subjected to short-path molecular distillation to separate the high-purity spiny-breasted frog oil main fraction. Step S5: In-situ reconstruction of functional greases: Lecithin and phytosterols were added to the main fraction of high-purity spiny-breasted frog oil obtained in step S4, and homogenization was carried out to obtain self-emulsifying functional spiny-breasted frog oil. Step S6: Full utilization of by-products: The aqueous phase and solid residue mixture obtained in step S3 were extracted, concentrated and dried to obtain spiny-breasted frog active peptide powder and calcium-phosphorus complex.

2. The extraction process for spiny-breasted frog oil using enzymatic methods according to claim 1, characterized in that, The directional maturation mentioned in step S1 specifically involves cooling the slurry to 0-4℃, maintaining a humidity of 80-90%, and allowing it to stand for 16-20 hours. The freeze-thaw cycle treatment in step S2 specifically involves cooling the directional matured slurry to -35 to -45°C for 100 to 140 minutes, then heating it to 0 to 4°C for 50 to 70 minutes, and repeating the above operation 2 to 4 times.

3. The extraction process for spiny-breasted frog oil using enzymatic methods according to claim 1, characterized in that, The mass ratio of the slurry to deionized water in step S2 is 0.4-0.6:

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4. The extraction process for spiny-breasted frog oil using enzymatic methods according to claim 1, characterized in that, The complex enzyme agent described in step S2 includes a 1,3-position specific lipase, a neutral protease, and a chitinase.

5. The extraction process for spiny-breasted frog oil using enzymatic methods according to claim 4, characterized in that, Based on the quality of the slurry, the amount of the compound enzyme system added is: 0.4%-0.6% 1,3-position specific lipase, 0.8%-1.2% neutral protease, and 0.08%-0.12% chitinase.

6. The extraction process for spiny-breasted frog oil using enzymatic methods according to claim 1, characterized in that, In the enzymatic hydrolysis reaction described in step S2, the reaction temperature is 45-55℃ and the reaction time is 160-200 min; The pH adjustment in step S2 specifically involves adding 0.5 mol / L citrate-sodium citrate buffer solution to adjust the pH to 6.4-6.

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7. The extraction process for spiny-breasted frog oil using enzymatic methods according to claim 1, characterized in that, The parameters of the pulsed ultrasonic field in step S2 are: frequency 35-45Hz, power density 45-55W / L, working mode is ultrasound for 4-6s, interval for 8-12s.

8. The extraction process for spiny-breasted frog oil using enzymatic methods according to claim 1, characterized in that, The ethanol-ammonium sulfate biphase extraction in step S3 is specifically as follows: add 85% ethanol aqueous solution to the oil-emulsion mixture, heat to 35-45℃, rotate at 100-200 rpm, stir for 15-25 min, add ammonium sulfate, let stand for 40-60 min, and take the alcohol phase after separation. The mass ratio of the oil-emulsion mixture, the ethanol-water solution, and the ammonium sulfate is 1:1.4-1.6:0.37-0.

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9. The extraction process for spiny-breasted frog oil using enzymatic methods according to claim 1, characterized in that, The short-range molecular distillation described in step S4 is a two-stage distillation. The first stage conditions are: evaporation temperature 110-130℃, vacuum degree 0.8-1.0Pa, and the second stage conditions are: evaporation temperature 170-190℃, vacuum degree 0.3-0.5Pa.

10. The extraction process for spiny-breasted frog oil using enzymatic methods according to claim 1, characterized in that, In step S5, the amount of lecithin added is 4%-6% of the mass of the main fraction of high-purity spiny-breasted frog oil, and the amount of phytosterol added is 1.5%-2.5%. The homogenization process described in step S5 is performed under the following conditions: nitrogen atmosphere, temperature 55-65℃, rotation speed 8000-12000 rpm, for 4-6 minutes.

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