A method for simultaneously and efficiently extracting crocodile peptides and crocodile oil

Abstract

This application discloses a method for simultaneously and efficiently extracting crocodile peptides and crocodile oil, relating to the field of bio-extraction technology. Using crocodile meat as raw material, the method involves sequentially heating and micro-pressure treatment to promote component dissolution; employing a multi-stage enzymatic hydrolysis process, sequentially using alkaline protease, neutral protease, and lipase, with calcium phosphate filtration aids and gradient addition of natural antioxidants during the hydrolysis process; subsequently, plate and frame filtration and static stratification are used to achieve crude separation of oil peptides; finally, the crocodile peptide solution undergoes multi-stage membrane filtration and spray drying, while the crude crocodile oil undergoes ultrasonic-assisted deodorization, ceramic membrane fine filtration, and gradient cooling phase change purification. After enzymatic hydrolysis and before filtration, a composite additive is added to the hydrolysate and stirred. This process achieves simultaneous and efficient extraction of crocodile peptides and oils, with no organic solvents throughout, significantly improving product yield, purity, and oxidative stability.

Description

Technical Field

[0001] This invention relates to the field of bio-extraction technology, and in particular to a method for simultaneously and efficiently extracting crocodile polypeptides and crocodile oil. Background Technology

[0002] Crocodile meat is rich in high-quality protein and functional oils, making it an ideal raw material for preparing bioactive peptides and nutritional oils. However, existing extraction processes have significant limitations: most adopt a single production model of "extracting peptides and discarding oil" or "extracting oil and discarding peptides," resulting in a raw material utilization rate of less than 50%, causing serious waste of resources and failing to achieve high-value utilization of by-products.

[0003] Furthermore, traditional processes generally face challenges in product quality and processing efficiency. Separate extraction requires two independent systems, resulting in complex processes and high energy consumption; during processing, oils are easily oxidized, leading to increased acid value and peroxide value, impaired peptide activity, and incomplete removal of odorous substances. Some processes rely on organic solvents, posing a risk of residue, while single antioxidants have limited effectiveness, contradicting the trend of green manufacturing. Existing technologies such as supercritical CO2 extraction can obtain low-acid-value oils, but they require sophisticated equipment, are expensive, and fail to fundamentally solve the problems of oil crystal structure regulation and long-term oxidative stability. Summary of the Invention

[0004] This application provides a method for simultaneously and efficiently extracting crocodile peptides and crocodile oil, solving the technical problems of low resource utilization, high energy consumption, easy oxidation of products, and strong fishy smell in existing crocodile processing technologies. Through innovative use of a series of continuous processes, including heated micro-pressure pretreatment, multi-stage enzymatic hydrolysis, calcium phosphate filtration, gradient addition of natural antioxidants, ultrasonic-assisted deodorization, and gradient cooling phase transition, the method achieves simultaneous and efficient extraction of crocodile peptides and crocodile oil from the same crocodile meat raw material. This process achieves simultaneous and efficient extraction of crocodile peptides and oils, without the use of organic solvents throughout, significantly improving product yield, purity, and oxidative stability.

[0005] This application provides a method for simultaneously and efficiently extracting crocodile polypeptides and crocodile oil, comprising the following steps: raw material processing: washing and cutting crocodile meat into pieces;

[0006] Heating and micro-pressure treatment: Mix crocodile meat and water at a mass ratio of 1:2, heat to 70-90℃, and then maintain the system temperature at 105℃ for 1 hour under micro-pressure conditions.

[0007] Cooling: Cooling the processed material;

[0008] Enzymatic hydrolysis: Alkaline protease hydrolysis, neutral protease hydrolysis, and lipase hydrolysis were performed sequentially.

[0009] Filtration: Add adsorbent and perform plate and frame filtration, then collect the filtrate;

[0010] Layering: Add rosemary extract to the filtrate, cool and allow to stand to separate the layers, obtaining an upper crude oil and a lower polypeptide liquid;

[0011] Peptide purification: The lower layer peptide solution is filtered through a membrane, concentrated, and dried to obtain crocodile peptide products;

[0012] Oil refining: The upper crude oil is deodorized, filtered, and subjected to gradient cooling to obtain refined crocodile oil products;

[0013] In this process, after enzymatic hydrolysis and before filtration, a composite additive is added to the enzymatic hydrolysate and stirred. The composite additive includes phospholipids, sucrose fatty acid esters with an HLB value of 13-16, and sucrose fatty acid esters with an HLB value of 5-9.

[0014] Furthermore, the addition of the composite additive adopts a step-by-step addition process, specifically including:

[0015] Add 40%-60% of the total amount of sucrose fatty acid esters with an HLB value of 13-16 to the enzymatic hydrolysate and stir at 300-400 rpm for 5-8 minutes.

[0016] Add the premix of phospholipids and all sucrose fatty acid esters with an HLB value of 5-9 to the enzymatic hydrolysate, increase the stirring speed to 400-500 rpm, and act for 15-20 minutes.

[0017] Add the remaining sucrose fatty acid ester with an HLB value of 13-16, adjust the stirring speed to 200-300 rpm, and let it work for 5-10 minutes.

[0018] Furthermore, the mass ratio of the phospholipid, the sucrose fatty acid ester with an HLB value of 13-16, and the sucrose fatty acid ester with an HLB value of 5-9 is 1 : (0.2-0.5) : (0.2-0.5).

[0019] Furthermore, the enzymatic hydrolysis treatment specifically includes:

[0020] First enzymatic hydrolysis: Adjust the pH of the material to 8.5-9.0 with calcium hydroxide, add 0.5%-2% of alkaline protease by the total mass of the material, and enzymatically hydrolyze at 45-55℃ for 0.5-2 hours, then boil to inactivate the enzyme;

[0021] Second enzymatic hydrolysis: Adjust the pH of the material to 7.0 with phosphoric acid, add 0.5%-4% of neutral protease by total mass of the material, and hydrolyze at 45-55℃ for 0.5-2 hours. This enzymatic hydrolysis does not inactivate the enzyme.

[0022] Third enzymatic hydrolysis: Add 0.1%-0.5% citric acid and 0.5%-5% lipase by weight of the material to the material, and hydrolyze at 45-55℃ for 0.5-4 hours, then inactivate the enzyme.

[0023] Furthermore, in the filtration step, the adsorbent is diatomaceous earth, and its addition amount is 3%-5% of the total mass of the enzymatic hydrolysate. The plate and frame filtration temperature is 60°C.

[0024] Furthermore, in the peptide refining step, membrane filtration is performed sequentially through membrane systems of 10000 Da and 200 Da; in the oil refining step, the deodorization is performed by ultrasonic deodorization under nitrogen protection, with conditions of frequency 10-20 kHz, power 150 W, and time 30-60 s; the gradient cooling is performed from 60℃ to 25℃ at a rate of 5℃ / h.

[0025] Furthermore, in the layering step and the oil refining step, the amount of rosemary extract added is 0.05%-0.1% of the total mass of the filtrate and 0.05%-0.1% of the total mass of the refined oil, respectively.

[0026] Furthermore, the phospholipid is food-grade soybean lecithin with a purity ≥95% and a particle size of 80-120 mesh; the sucrose fatty acid ester is food-grade with a purity ≥98%.

[0027] One or more technical solutions provided in the embodiments of this application have at least the following technical effects or advantages:

[0028] This process simultaneously extracts crocodile oil and crocodile peptides from crocodile meat, reducing energy consumption. It is simple, easy to operate, and highly feasible, aligning with sustainable development principles. Furthermore, it uses natural ingredients without organic solvents. This method achieves efficient utilization of raw materials, reducing resource waste. It overcomes the limitations of existing processes that either extract peptides and discard oil or extract oil and discard peptides, simultaneously extracting crocodile polypeptides and crocodile oil through a continuous process. This achieves full utilization of crocodile meat raw materials, avoiding resource waste and improving the overall utilization rate of raw materials.

[0029] By introducing phospholipids and sucrose fatty acid esters, this study addresses the technical problems of low oil-peptide separation efficiency, low product yield, and easy oxidation of oils caused by the stubborn emulsion system formed by amphiphilic substances such as proteins and peptides during enzymatic hydrolysis. Phospholipids and sucrose fatty acid esters interact at the molecular level at the interface, jointly constructing a composite interface film with a more disordered structure and significantly reduced mechanical strength. This composite interface film greatly promotes the coalescence kinetics of oil droplets, thereby significantly improving the oil-peptide separation efficiency. In addition, this composite interface structure exhibits stronger affinity and fixation ability for lipid-soluble antioxidant molecules, forming a denser antioxidant protective barrier on the oil surface, thus synergistically improving the oxidative stability of oils from both physical barrier and chemical removal dimensions.

[0030] By introducing sucrose fatty acid esters with specific HLB value combinations and employing a stepwise addition process, a spatiotemporally ordered interface regulation system is formed with phospholipids. Based on the functional gradient design and temporal synergistic effect of each component, the technical challenge of synergistically improving oil-peptide separation efficiency and product oxidative stability is solved, thereby achieving further improvement in process efficiency and product quality. Detailed Implementation

[0031] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention pertains; the terminology used herein in the description of the invention is for the purpose of describing particular embodiments only and is not intended to limit the invention; the term "and / or" as used herein includes any and all combinations of one or more of the associated listed items.

[0032] Example 1: A method for simultaneously and efficiently extracting crocodile polypeptides and crocodile oil, specifically including the following steps:

[0033] 1. Raw material processing: Select legally farmed crocodile meat, clean it, and cut it into pieces for later use;

[0034] 2. Heating and micro-pressure treatment: Mix crocodile meat and water in a 1:2 ratio, heat to 70-90℃, and then micro-pressure to 105℃ for 1 hour to promote the dissolution of components;

[0035] 3. Cooling: Cool the material to 20℃;

[0036] 4. Enzymatic hydrolysis:

[0037] Adjust the pH to 8.5-9.0, add 0.5%-2% alkaline protease, hydrolyze at 45-55℃ for 0.5-2 hours, and then boil to inactivate the enzyme;

[0038] Adjust the pH to 7.0, add 0.5%-4% neutral protease, and enzymatically hydrolyze at 45-55℃ for 0.5-2 hours without inactivating the enzyme;

[0039] Add 0.1%-0.5% citric acid and 0.5%-5% lipase, and enzymatically hydrolyze for 0.5-4 hours to inactivate the enzyme;

[0040] 5. Filtration: Add 3%-5% diatomaceous earth, stir, and then filter at 60℃ using a plate and frame filter.

[0041] 6. Layer separation: Add 0.05%-0.1% rosemary extract to the filtrate, cool to 30℃ and allow to stand to separate into layers. The upper layer is crude oil and the lower layer is polypeptides.

[0042] 7. Peptide purification: The filtrate is filtered through 10000Da and 200Da membranes, concentrated to a concentration of 40%-60%, and then spray-dried.

[0043] 8. Oil refining: The crude oil is subjected to nitrogen protection, ultrasonic deodorization (10-20kHz, 150W, 30-60s), filtration through a 0.2μm ceramic membrane, and then rosemary extract is added before gradient cooling (from 60℃ to 25℃ at 5℃ / h) to obtain refined oil.

[0044] Experiments were conducted on the technical solution of this embodiment:

[0045] Case 1:

[0046] 1.1 Raw material processing: Select legally farmed crocodile meat, remove impurities, wash it thoroughly with clean water, and cut it into chunks for later use;

[0047] 1.2 Heating and micro-pressure treatment: Place the chopped crocodile meat pieces into the extraction container, add water at a mass ratio of crocodile meat to water of 1:2, then heat the temperature inside the extraction container to 85℃, and then perform micro-pressure treatment (the temperature corresponding to the micro-pressure is 105℃), and maintain it for 1 hour;

[0048] 1.3 First enzymatic hydrolysis: Cool the material treated in step 1.2 to 20℃, add calcium hydroxide to adjust the pH value of the material to 8.5-9.0; then control the temperature of the material at 45-55℃, add 1% (mass ratio) alkaline protease for enzymatic hydrolysis for 2 hours; after the enzymatic hydrolysis is completed, boil the material to inactivate the enzyme.

[0049] 1.4 Second enzymatic hydrolysis: Adjust the pH of the above materials to 7.0 with phosphoric acid; control the material temperature at 45-55℃, add 2% (mass ratio) neutral protease and perform enzymatic hydrolysis for 1 hour; no enzyme inactivation treatment is performed after this enzymatic hydrolysis;

[0050] 1.5 Third enzymatic hydrolysis: Add 0.3% (by mass) citric acid and 1% (by mass) lipase to the material from step 1.4, and perform enzymatic hydrolysis for 2 hours; after the enzymatic hydrolysis is completed, perform enzyme inactivation treatment;

[0051] 1.6 Adsorption and Filtration: Add 5% (mass ratio) of diatomaceous earth to the enzymatic hydrolysate, stir well, and after 20 minutes of action, perform plate and frame filtration at 60℃ and collect the filtrate.

[0052] 1.7 Adding antioxidants and separation: Add 0.05% (by mass) of rosemary extract to the filtrate collected in step 1.6, stir well, cool to 30°C, and allow to stand for separation;

[0053] 1.8 Product separation: Collect the liquid after separation. The upper layer is crude crocodile oil and the lower layer is crocodile polypeptide liquid. During this process, food-grade nitrogen gas (flow rate 5~10 L / min) is introduced to create a slightly positive pressure environment to isolate the air.

[0054] 1.9 Refining of Crocodile Peptides: The lower layer of crocodile peptide liquid obtained in step 1.8 is subjected to multiple membrane filtrations in sequence. First, it is passed through a 10000 Da water-soluble filter membrane, and then through a 200 Da water-soluble filter membrane to remove free amino acids. Then, the resulting small molecule peptide filtrate is concentrated to a solid content of 40%-60% using a membrane high-efficiency concentration device. Finally, it is spray-dried to obtain the crocodile small molecule peptide product.

[0055] 1.10 Deodorization of crocodile crude oil: The upper layer of crocodile crude oil obtained in step 1.8 was deodorized by ultrasound under the protection of food-grade nitrogen (flow rate 5~10 L / min) (conditions: 15kHz, 150W, 30 seconds per ultrasound, repeated 5 times, with 30-second intervals between each ultrasound); after deodorization, the oil was rapidly cooled to below 60℃.

[0056] 1.11 Crocodile oil filtration: The deodorized oil obtained in step 1.10 is filtered through a 0.2μm ceramic membrane to ensure the oil is clear;

[0057] 1.12 Refining of crocodile oil: Add 0.05% (by mass) of rosemary extract to the refined oil obtained in step 1.11, stir to dissolve, and then gradually reduce the oil temperature from 60℃ to 25℃ at a cooling rate of 5℃ / h to obtain the refined crocodile oil product.

[0058] Case 2 (producing only crocodile peptides);

[0059] The implementation steps in this case are exactly the same as steps 1.1 to 1.9 in Case 1, but in the production process, the separated oil is treated as waste and is not collected or refined.

[0060] Case 3 (Produces only crocodile oil);

[0061] The implementation steps in this case are the same as steps 1.1 to 1.7 in Case 1. Subsequently, in step 1.13, after stratification, only the upper oil layer is collected, and the lower water layer (i.e., crocodile polypeptide liquid) is removed. The subsequent steps are the same as steps 1.10 to 1.12 in Case 1, where the collected crude oil is deodorized, finely filtered, and refined to obtain refined crocodile oil.

[0062] Case 4 (Micro-pressure treatment without heating);

[0063] The implementation steps in this case are basically the same as in Case 1, except that the heating and micro-pressure treatment in step 1.2 is omitted.

[0064] Case 5 (Continuous production system for removing calcium phosphate).

[0065] The implementation steps in this case are basically the same as in Case 1. The difference is that when adjusting the pH value in steps 1.3 and 1.4, calcium hydroxide and phosphoric acid are not used, but sodium hydroxide and phosphoric acid are used instead.

[0066] Case 6 (No citric acid added during continuous production);

[0067] The implementation steps in this case are the same as steps 1.1 to 1.4 in Case 1. Subsequently, in step 1.14, without adding citric acid, 1% lipase is directly added for enzymatic hydrolysis for 0.5-4 hours, and the enzyme is inactivated after hydrolysis. Subsequent steps are the same as steps 1.6 to 1.12 in Case 1.

[0068] Case 7 (Continuous production without rosemary extract);

[0069] The implementation steps in this case are basically the same as in Case 1, except that rosemary extract is not added in steps 1.7 and 1.12.

[0070] Case 8 (Continuous production with simultaneous addition of antioxidants);

[0071] The implementation steps in this case are basically the same as in Case 1. The difference is that in steps 1.5, 1.7 and 1.12, 0.3% citric acid and 0.05% rosemary extract are added simultaneously as antioxidants.

[0072] Case 9 (Continuous production gradient without rosemary extract);

[0073] The implementation steps in this case are basically the same as in Case 1, except that in step 1.12, rosemary extract is no longer added.

[0074] Case 10 (Continuous production of crocodile oil without gradient temperature control).

[0075] The implementation steps in this case are basically the same as in Case 1. The difference is that in step 1.12, the gradient cooling process is canceled, and the refined oil is directly cooled from 60℃ to 25℃.

[0076] Case 11 (Continuous production of crocodile oil without ultrasonic-assisted deodorization);

[0077] The implementation steps in this case are basically the same as in Case 1, except that in step 1.10, the ultrasonic-assisted deodorization step is cancelled, and the crude crocodile oil is directly cooled.

[0078] Performance testing was performed on the above cases:

[0079] Experiment 1. Advantages of continuous production for the extraction of crocodile polypeptides and crocodile oil;

[0080] The yields of crocodile polypeptides and crocodile oil in Cases 1-3 were measured respectively, and the results are shown in Table 1 below.

[0081] Crocodile polypeptide yield (%) = (mass of dried polypeptide / total mass of protein in raw material) × 100%;

[0082] Crocodile oil yield (%) = (weight of refined oil / weight of total oil in raw materials) × 100%;

[0083] Polypeptide content (high performance liquid chromatography) and acid value were determined according to GB5009.229-2016 "Determination of Acid Value in Food" and "Determination of Peroxide Value in Food".

[0084] Table 1. Comparison of preparation of crocodile peptides and crocodile oil in Cases 1-3

[0085]

[0086] Experimental Results: Case 1 showed an 18% increase in peptide yield compared to Case 2, with higher product quality and content. Compared to Case 3, Case 1 showed a 15% increase in crocodile oil yield, and the quality remained unchanged according to conventional oil quality indicators such as acid value and peroxide value. Cases 2 and 3, due to the discarding of one component, had raw material utilization rates only 50%-60% of Case 1. Compared to Cases 2 plus Case 3, Case 1 showed a 16.2% decrease in energy consumption and an 85% reduction in processing time.

[0087] Experiment 2: Effect of micro-pressure treatment on yield;

[0088] By measuring the peptide yield and crocodile oil yield in Cases 1 and 4, it was found that micro-pressure treatment of the raw materials improved the yield of both peptides and oils, with a 10% increase in peptide yield and an 8% increase in crocodile oil yield. The test results are shown in Table 2 below.

[0089] Table 2. Advantages of micro-compression treatment of raw materials (comparison of Case 1 and Case 4)

[0090]

[0091] Experiment 3: Verification of the effects of gradient addition of natural antioxidants;

[0092] The gradient synergistic addition of citric acid and rosemary extract enhanced the antioxidant properties, stability, and retention of active ingredients in crocodile oil. The advantages of gradient synergistic addition were verified by comparing key indicators in Case 1 (gradual addition of citric acid and rosemary), Case 6 (no citric acid), Case 7 (no rosemary), Case 8 (simultaneous addition of both), and Case 9 (omitted rosemary). The test results are shown in Table 3 below.

[0093] Table 3. Effects of Gradient Addition of Natural Antioxidants

[0094]

[0095] (The color difference (ΔE) after 7 days of accelerated storage at 60℃ can be measured using a colorimeter, and the retention rate of fat-soluble vitamins (A, E) can be measured using high performance liquid chromatography (HPLC).)

[0096] Case 1, through the gradient addition of citric acid and rosemary extract, outperformed other cases in terms of peroxide value, acid value control, and retention of active ingredients, demonstrating the advantages of synergistic effects and gradient addition.

[0097] Experiment 4: Verify the importance of ultrasonic deodorization auxiliary process;

[0098] To verify the effect of ultrasound-assisted removal of crocodile oil odor and retention of antioxidant active ingredients, the differences between Case 1 (ultrasound-assisted deodorization) and Case 11 (no ultrasound deodorization) were compared.

[0099] Testing indicators:

[0100] Fishy odor removal rate: The residual amount of volatile sulfur-containing compounds (such as methanethiol and dimethyl sulfide) was determined by gas chromatography-mass spectrometry (GC-MS), and the removal rate was calculated (removal rate = (content before treatment - content after treatment) / content before treatment × 100%).

[0101] Sensory evaluation: Ten professional evaluators blindly evaluated the fishy smell intensity of the samples using a 9-point scale (1 point = no fishy smell, 9 points = extremely strong fishy smell), and the average value was taken.

[0102] Content of fat-soluble vitamins: The content of vitamins A and E was determined by high performance liquid chromatography (HPLC);

[0103] DPPH free radical scavenging rate: determined by spectrophotometry, reflecting antioxidant activity. The results are shown in Table 4 below.

[0104] Table 4. Effect of ultrasonic deodorization process on activity

[0105]

[0106] Case 1, due to the use of ultrasound assistance, achieved a significantly higher removal rate of volatile sulfur compounds than Case 11, a lower sensory score (weaker fishy smell), and more complete retention of vitamins A and E, as well as a higher DPPH removal rate. This demonstrates the advantage of ultrasound assistance in enhancing deodorization while promoting the retention of antioxidant components, with a reduction in fishy smell of over 50%.

[0107] Experiment 5: Effect of gradient cooling phase transition on the stability of oils and fats;

[0108] Case 1 (Gradual Cooling): After adding 0.05% rosemary extract to the refined oil, the temperature was gradually reduced from 60℃ to 25℃ at a rate of 5℃ / h. Case 10 (Direct Cooling): After adding the same dose of rosemary extract to the refined oil, the temperature was directly reduced from 60℃ to 25℃ (without gradient). This verified the effect of gradient cooling phase transition (gradually reducing from 60℃ to 25℃ at a rate of 5℃ / h) on improving the crystal structure stability and oxidation resistance of crocodile oil.

[0109] Measurement methods: Crystal structure: X-ray diffraction (XRD) was used to determine the grain size and distribution of oil crystals and characterize crystal stability;

[0110] Oxidative stability: The peroxide value (meq / kg) and acid value (mg KOH / g) were determined after 0, 30, and 60 days of storage at room temperature (25℃).

[0111] Unsaturated fatty acid exposure area: determined by low-temperature nuclear magnetic resonance (NMR), reflecting oxidation risk;

[0112] Sensory stability: Observe the change in oil color (color difference ΔE) during storage. The test results are shown in Table 5 below;

[0113] Table 5. Effects of gradient cooling phase transition on oil stability

[0114]

[0115] Based on the initial acid value of 3.5 mg / g and peroxide value of 4.9 mmol / kg in Case 1 (60 days ago), after 60 days of storage, Case 1, due to the use of gradient cooling, showed a smaller increase in acid value and peroxide value (0.3 mg / g and 0.4 mmol / kg, respectively), while Case 10 (direct cooling) showed a more significant increase (1.0 mg / g and 1.9 mmol / kg, respectively). Gradient cooling can reduce the risk of unsaturated fatty acid oxidation and significantly improve the stability of oils. The phase transition of gradient cooling makes oils more stable.

[0116] Experiment 6: Effect of the calcium phosphate system on the process;

[0117] Case 1: During the enzymatic hydrolysis stage, the pH was adjusted to 8.5-9.0 with calcium hydroxide, and then adjusted to 7.0 with phosphoric acid (forming a calcium phosphate system). Case 5: During the enzymatic hydrolysis stage, the pH was adjusted to 8.5-9.0 with sodium hydroxide, and then adjusted to 7.0 with phosphoric acid (a calcium phosphate-free system). Filtration efficiency: The plate and frame filtration time (60℃) was measured (min); Product clarity: The transmittance (%) of the peptide solution at 600nm was measured using a UV-Vis spectrophotometer. Higher transmittance indicates better clarity.

[0118] Case 1, using a calcium phosphate system, significantly shortened the filtration time, resulting in higher transmittance of the peptide solution (better clarity) and a 4.2% increase in yield compared to Case 5 (within the 3%-5% range). The test results are shown in Table 6 below.

[0119] Table 6. Process Influence of Calcium Phosphate System

[0120]

[0121] The technical solutions described in the embodiments of this application have at least the following technical effects or advantages:

[0122] The purpose of this embodiment is to provide a continuous and efficient method for simultaneously extracting crocodile peptides and crocodile oil, and its applications. The obtained crocodile peptides and crocodile oil have advantages such as high purity, high stability, low fishy smell, and strong anti-inflammatory activity. Furthermore, this process achieves simultaneous extraction of crocodile oil and crocodile peptides from crocodile meat, reducing energy consumption, simplifying the process, making it easy to operate, highly feasible, and in line with sustainable development. It also eliminates the use of organic solvents and utilizes natural components. This method achieves efficient utilization of raw materials and reduces resource waste. It overcomes the limitations of existing processes that either extract peptides and discard oil or extract oil and discard peptides, achieving full utilization of crocodile meat raw materials through continuous simultaneous extraction of crocodile peptides and crocodile oil, avoiding resource waste, and improving the comprehensive utilization rate of raw materials.

[0123] The product boasts high purity and improved yield; crocodile oil exhibits low acid value and peroxide value, retains its active ingredients intact, and significantly enhances stability; crocodile peptides are free of free amino acids, ensuring stable quality and outstanding anti-inflammatory and antioxidant properties, particularly effective in improving diabetic foot inflammation. Through ultrasound-assisted deodorization and gradient cooling phase transition technologies, the product's fishy odor is effectively reduced, and the oil forms a more stable crystal structure, minimizing the risk of unsaturated fatty acid oxidation and extending the product's shelf life.

[0124] The entire process employs green technology, eliminating the use of organic solvents and avoiding residue risks. It selects natural ingredients such as citric acid, rosemary extract, and tea polyphenols as antioxidants; when used in combination, their antioxidant effect is superior to that of a single antioxidant, meeting "clean label" requirements and aligning with the trend of green biomanufacturing. The gradient synergistic addition of these compound natural antioxidants overcomes the limitations of single antioxidants, employing a gradient addition of citric acid and plant polyphenols. The synergistic effect of both enhances the antioxidant effect compared to using either alone, and both are natural ingredients, complying with green technology and green labeling requirements.

[0125] By combining inert gas microenvironment protection with low-temperature phase change technology, physical protection is integrated with the phase change characteristics of oils, reducing oxygen contact during processing, improving oil quality, and lowering the risk of oxidation. Enzymatic hydrolysis parameters are adjusted to reduce easily oxidizable impurities in the oil, and ultrasound-assisted deodorization is enhanced while promoting the retention of antioxidants. Existing processes do not use microwave deodorization to remove the fishy smell from crocodile oil. A gradient cooling phase change is employed after refining, causing the oil to form a more stable crystal structure and reducing the exposed area of ​​unsaturated fatty acids. This results in more stable and higher-quality crocodile oil.

[0126] Compared to traditional processes, the resulting crocodile peptides have higher anti-inflammatory properties, and the crocodile oil has lower acid value and peroxide value, resulting in stronger antioxidant properties.

[0127] The obtained crocodile polypeptides and crocodile oil can be directly applied to the fields of biological products, nutritional health foods, and cosmetics. The complex formed by the two has outstanding application potential in improving specific inflammations (such as diabetic foot) due to its high anti-inflammatory properties, providing a new path for the high-value utilization of crocodile resources.

[0128] Example 2: Addressing the resource waste, high energy consumption, easy oxidation, and strong fishy odor caused by either peptide extraction and oil discarding or oil extraction and peptide discarding in existing crocodile processing, Example 1 provides a method for simultaneously and efficiently extracting crocodile peptides and crocodile oil. Through processes such as heated micro-pressure treatment, multi-stage enzymatic hydrolysis, calcium phosphate filtration, gradient antioxidant effects from citric acid and rosemary extracts, ultrasonic-assisted deodorization, and gradient cooling phase transition, continuous and simultaneous extraction of crocodile peptides and oil is achieved. This ultimately improves the yield and purity of both peptides and oil, reduces energy consumption, shortens processing time, and results in a product with low fishy odor, high stability, significant antioxidant and anti-inflammatory activity, and no organic solvents throughout the process, meeting green manufacturing and clean label requirements. Further improvements are made based on Example 1 to further enhance the overall performance of the product.

[0129] A composite additive is added after the enzymatic hydrolysis step, the composite additive including phospholipids and sucrose fatty acid esters;

[0130] The phospholipid is food-grade soybean lecithin with a purity of ≥95%; the addition amount is 0.1%-0.5% based on the total mass of the enzymatic hydrolysate, with a preferred range of 0.2%-0.3%; the particle size is 80-120 mesh.

[0131] The sucrose fatty acid ester has an HLB value of 10-16 (preferably 13-14), an addition amount of 20%-50% based on the phospholipid content, a particle size of 100-150 mesh, and a purity of food grade ≥98%.

[0132] The compound additive is added by using a premixed pulping process: phospholipids and sucrose fatty acid esters are dry-mixed, then mixed with warm water to form a pulp, and then fed into the feed. The feeding temperature is 45-55℃, and the stirring speed is 300-500 rpm.

[0133] The specific steps for adding the compound additive are as follows:

[0134] Weigh out phospholipids and sucrose fatty acid esters in proportion, and mechanically mix them in a dry container for 10-15 minutes at room temperature (20-25℃) and stirring speed (100-200 rpm).

[0135] The dry-mixed composite additive is mixed with warm water at a mass ratio of 1:3 to 1:5, and pre-emulsified using a high-speed shear emulsifier.

[0136] The parameters are as follows: water temperature: 50-55℃; shearing speed: 8000-12000 rpm; time: 3-5 minutes.

[0137] The pre-prepared slurry is slowly added to the enzymatic hydrolysate while being stirred.

[0138] The enzyme hydrolysate temperature is 45-55℃; the stirring speed is 300-500 rpm; the feeding time is 5-8 minutes; and the reaction time is 20-30 minutes.

[0139] Based on Example 1, this embodiment conducts an experiment to verify the effect of adding phospholipids (PL) and sucrose fatty acid esters (SE) on improving product yield and quality, based on the process of Example 1.

[0140] Experimental grouping and parameter design:

[0141] All experimental groups followed the baseline process of Case 1 in Example 1, with the only difference being the type and proportion of additives after enzymatic hydrolysis and before filtration. A total of 6 experimental groups and 1 control group were designed. The experimental groups are shown in Table 7 below;

[0142] Table 7

[0143]

[0144] All additives are added after dry premixing and wet pulping.

[0145] Pulping parameters: The composite additive is mixed with warm water (50-55℃) at a mass ratio of 1:4;

[0146] Shearing parameters: High-speed shearing machine, 10,000 rpm, processing time 3 minutes;

[0147] Feeding and reaction parameters: Feed the material into the enzymatic hydrolysate at 45-55℃ with stirring at 400 rpm for a total reaction time of 25 minutes;

[0148] Performance testing metrics and methods: Based on the testing content of Example 1, the following metrics were tested:

[0149] Plate and frame filtration time (min): reflects the demulsification effect and the clarity of the system;

[0150] Settling time (min): Take a uniform volume of the filtrate after filtration into a stoppered graduated cylinder and place it in a 30°C constant temperature water bath to stand. Record the time required from the start of settling until the interface between the oil phase and the aqueous phase (peptide solution) is clear and the volume of the interface layer no longer changes significantly.

[0151] Crocodile polypeptide yield (g / 100g): Calculation method is the same as in Example 1;

[0152] Crocodile oil yield (mg / g): Calculation method is the same as in Example 1;

[0153] Vitamin E retention rate (60°C, 7 days) (%): Refined crocodile oil samples were placed in a 60°C constant temperature oven for accelerated oxidation experiments and stored for 7 days. The vitamin E content in the oil was determined by high performance liquid chromatography (HPLC) before storage (0 days) and after storage (7 days). The calculation formula is: Vitamin E retention rate (%) = (Vitamin E content after 7 days of storage / Vitamin E content after 0 days of storage) × 100%.

[0154] Crocodile oil peroxide value (mmol / kg): reflects the initial degree of oxidation of the oil;

[0155] The test results are shown in Table 8 below;

[0156] Table 8

[0157]

[0158] Experimental group F, where phospholipids were prepared alone, showed inferior dispersion and final performance compared to groups prepared with SE. This experiment, through systematic comparison, demonstrates that introducing a phospholipid-sucrose fatty acid ester composite system (using the process parameters of Example 2) based on Example 1 can improve process efficiency and enhance product yield and quality through synergistic effects. Furthermore, PL 0.3% and SE (HLB~14) 0.09% represent the optimal ratio under the experimental conditions.

[0159] The technical solutions described in the embodiments of this application have at least the following technical effects or advantages:

[0160] By introducing phospholipids and sucrose fatty acid esters, technical problems such as low oil-peptide separation efficiency, low product yield, and easy oxidation of oils caused by the stubborn emulsion system formed by the presence of amphiphilic substances such as proteins and peptides during enzymatic hydrolysis are solved.

[0161] Phospholipids, as amphiphilic molecules, can competitively adsorb at the oil-water interface, effectively replacing and disrupting the original interfacial film composed of proteins and peptides. This significantly reduces interfacial stability, promotes the coalescence of dispersed oil droplets, and achieves efficient demulsification. Simultaneously, the oriented layer formed by phospholipids at the interface possesses certain antioxidant functions and can enhance its local concentration and antioxidant efficacy in the interfacial region through intermolecular interactions with antioxidants such as rosemary extract.

[0162] The function of sucrose fatty acid esters is designed based on their hydrophilic-lipophilic balance (HLB) values. Sucrose esters with high HLB values ​​can rapidly reduce interfacial tension and preferentially adsorb onto the interface, creating favorable conditions for subsequent interfacial processes. The micellar structure formed by their molecules in the aqueous phase can effectively disperse and transport phospholipids, preventing aggregation during feeding and ensuring the full realization of their biointerfacial activity.

[0163] The synergistic effect of phospholipids and sucrose fatty acid esters lies in their molecular-level interactions at the interface, jointly constructing a composite interfacial film with a more disordered structure and significantly reduced mechanical strength. This composite interfacial film greatly promotes the coalescence kinetics of oil droplets, thereby significantly improving oil-peptide separation efficiency. Furthermore, this composite interfacial structure exhibits stronger affinity and immobilization ability for lipid-soluble antioxidant molecules, forming a denser antioxidant protective barrier on the oil surface, synergistically enhancing the oxidative stability of oils from both physical barrier and chemical scavenging perspectives.

[0164] Example 3: Example 2, by introducing phospholipids and sucrose fatty acid esters and utilizing their synergistic effect, solved the problems of low separation efficiency, low yield, and poor oxidative stability through a series of coherent mechanisms, including competitive interfacial adsorption, composite interfacial membrane construction, and synergistic antioxidant effects, achieving simultaneous improvement in process efficiency and product quality. To further improve the overall performance of the product, further improvements were made based on Example 2.

[0165] The sucrose fatty acid esters include high HLB value sucrose fatty acid esters and low HLB value sucrose fatty acid esters. The high HLB value sucrose fatty acid esters have an HLB value range of 13-16 and an addition amount of 20%-50% based on the phospholipid content.

[0166] Low HLB value sucrose fatty acid esters: HLB value range: 5-9; addition amount: 20%-50% based on phospholipid content.

[0167] Phospholipids, high HLB sucrose esters, and low HLB sucrose esters were premixed in a ratio of PL:SE-H:SE-L = 1:(0.2-0.5):(0.2-0.5).

[0168] The addition method adopts a step-by-step process, and the specific steps are as follows:

[0169] Weigh out phospholipids (PL), high HLB sucrose esters (SE-H), and low HLB sucrose esters (SE-L) according to the above preferred ratio; divide SE-H into two parts: SE-H1 (accounting for 60% of the total SE-H) and SE-H2 (accounting for 40% of the total SE-H); dry premix all PL and all SE-L to obtain PL-SE-L premix;

[0170] SE-H1 was slowly added to the enzymatic hydrolysate at 45-55℃, and stirred at 300-400 rpm for 5-8 minutes. During this stage, the rapid diffusion and adsorption capacity of the high HLB value sucrose ester was utilized to preferentially occupy the oil-water interface, achieving an initial reduction and activation of the system's interfacial tension.

[0171] Add the PL-SE-L premix to the pre-activated system, increase the stirring speed to 400-500 rpm, and act for 15-20 minutes. During this stage, phospholipids are rapidly adsorbed in the favorable environment created by SE-H, and synergistically with low HLB sucrose esters that can penetrate deep into the interfacial membrane to jointly construct the unstable composite interfacial membrane main structure;

[0172] Add the remaining SE-H2, adjust the stirring speed to 200-300 rpm, and react for 5-10 minutes. This stage utilizes high HLB sucrose esters to further optimize interface coverage, repair potential interface defects, and enhance the dispersion stability of the system, preparing for subsequent separation steps.

[0173] Based on the experimental group D of Example 2, this embodiment conducts an experiment as experimental group G. Based on the experimental group D of Example 2 (PL 0.3% + SE 0.09%, HLB=14), the improvement effect of introducing sucrose fatty acid esters with different HLB values ​​and the stepwise addition process in Example 3 is verified.

[0174] The difference between experimental group G and experimental group D is that they include high HLB value sucrose fatty acid esters (SE-H) and low HLB value sucrose fatty acid esters (SE-L), and are added in a step-by-step manner as described in this embodiment: The experimental groups are shown in Table 9 below;

[0175] Table 9

[0176]

[0177] Key process parameters;

[0178] The step-by-step addition process is the same as before (first stage: 60% SE-H, second stage: PL + all SE-L, third stage: 40% SE-H);

[0179] The total duration of action is uniformly set at 34 minutes;

[0180] Other process parameters are the same as in Example 2; the test results are shown in Table 10 below.

[0181] Table 10

[0182]

[0183] Experimental results show that different ratios achieve targeted regulation of interfacial performance through differentiated cooperation at the molecular level: the low HLB-dominant ratio (2:3) focuses on building a dense interfacial barrier, significantly enhancing the product's oxidative stability; the high HLB-dominant ratio (2:1) strengthens the rapid interface occupation ability and improves the initial separation efficiency; while the balanced ratio (1:1) can maximize the synergistic effect under the stepwise addition process, achieving simultaneous optimization of separation efficiency and product stability, with a 23.1% increase in demulsification rate and simultaneous enhancement of antioxidant performance, demonstrating the best comprehensive technical and economic benefits.

[0184] The technical solutions described in the embodiments of this application have at least the following technical effects or advantages:

[0185] By introducing high and low HLB value sucrose fatty acid esters and using a stepwise addition process, a composite interface regulation system is constructed to solve the technical problem that single HLB value surfactants are difficult to balance rapid demulsification and long-term stability in complex emulsion systems. In particular, it addresses the bottleneck of separation efficiency caused by excessively high interfacial film strength during oil-peptide separation, as well as the defects of conventional antioxidant systems such as insufficient oxidative stability caused by uneven distribution at the interface.

[0186] High HLB-value sucrose fatty acid esters are added in the first stage of the stepwise addition process. Their strong hydrophilicity rapidly reduces the interfacial tension of the system, enabling preferential occupation and pre-activation of the oil-water interface, creating a thermodynamic advantage for the interfacial adsorption of subsequent components. Low HLB-value sucrose fatty acid esters are added together with phospholipids in the second stage. Their strong hydrophobic properties allow them to penetrate the pretreated interfacial layer, deeply disrupting the structural integrity of the original interfacial film and interacting specifically with the hydrophobic regions of the phospholipids. In the third stage, the remaining high HLB-value sucrose fatty acid esters are added again to further optimize interfacial coverage, fill any potential interfacial defects, and, through intermolecular forces, work with the already adsorbed phospholipid-low HLB-value sucrose ester system to complete the final stable construction of the interfacial film, thereby improving the uniformity and integrity of the interfacial film.

[0187] High and low HLB values ​​complement each other during interfacial adsorption through the gradient difference in hydrophilic-lipophilic properties. The high HLB value component achieves rapid interfacial coverage, while the low HLB value component completes deep interfacial reconstruction, jointly constructing a composite interfacial membrane with structural heterogeneity. This dual HLB value system synergizes with phospholipids, which serve as the interfacial architecture matrix. Phospholipids undergo molecular assembly with the two sucrose esters adsorbed sequentially at the interface, forming a composite membrane structure with reduced mechanical stability but increased interfacial coverage.

[0188] The stepwise addition process aims to avoid competitive inhibition between components with different HLB values ​​during interfacial adsorption by controlling the timing of functional component addition. This achieves programmed and optimized interfacial regulation, ensuring that each component performs its stage-specific function during interfacial formation, thereby optimizing synergistic effects. High HLB value components are preferentially adsorbed in the first stage to achieve pre-activation and initial occupation of the interface. Low HLB value components are added together with phospholipids in the second stage to complete deep penetration and interfacial structure disruption. The addition of high HLB value components in the third stage further enhances interfacial coverage. This timing design, through functional gradient complementarity and ordered molecular assembly, forms a composite interfacial membrane with improved structural heterogeneity and optimized mechanical stability.

[0189] Through a multi-level interface regulation mechanism, the stability of the oil-peptide emulsion system is reduced, and the efficiency of the separation process is improved by more than 20%. At the same time, the formed composite interface membrane can more effectively enrich and fix antioxidant molecules, improve the oil oxidation stability index by more than 15%, and achieve a breakthrough in product yield and quality simultaneously.

[0190] By introducing sucrose fatty acid esters with specific HLB value combinations and employing a stepwise addition process, a spatiotemporally ordered interface regulation system is formed with phospholipids. Based on the functional gradient design and temporal synergistic effect of each component, the technical challenge of synergistically improving oil-peptide separation efficiency and product oxidative stability is solved, thereby achieving further improvement in process efficiency and product quality.

[0191] The above description is merely a preferred embodiment of the present invention and is not intended to limit the invention. For those skilled in the art, the present invention can have various modifications and variations. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of the present invention should be included within the scope of protection of the present invention.

Claims

1. A method for simultaneously efficiently extracting a crocodile polypeptide and crocodile oil, characterized by, The method comprises the following steps: Raw material processing: clean and cut the alligator meat; Warm micro-pressure treatment: mix the alligator meat with water at a mass ratio of 1:2, warm to 70-90℃, and make the system temperature reach 105℃ under micro-pressure for 1 hour; Cooling: cool the treated material; Enzymatic hydrolysis: sequentially perform alkaline protease enzymatic hydrolysis, neutral protease enzymatic hydrolysis, and lipase enzymatic hydrolysis; The first enzymatic hydrolysis: cool the material treated in step 1.2 to 20℃, add calcium hydroxide to adjust the pH of the material to 8.5-9.0; then control the temperature of the material at 45-55℃, and add 1% (mass ratio) of alkaline protease for enzymatic hydrolysis for 2 hours; after the enzymatic hydrolysis is completed, boil the material to inactivate the enzyme; The second enzymatic hydrolysis: adjust the pH of the material after the first enzymatic hydrolysis to 7.0 with phosphoric acid; control the temperature of the material at 45-55℃, and add 2% (mass ratio) of neutral protease for enzymatic hydrolysis for 1 hour; after the enzymatic hydrolysis is completed, do not perform inactivation treatment; The third enzymatic hydrolysis: add 0.3% (mass ratio) of citric acid and 1% (mass ratio) of lipase to the material after the second enzymatic hydrolysis, and perform enzymatic hydrolysis for 2 hours; after the enzymatic hydrolysis is completed, perform inactivation treatment; Filtration: add an adsorbent to perform plate and frame filtration to collect the filtrate; Layering: add rosemary extract to the filtrate, cool and stand to separate layers, and separate to obtain upper crude oil and lower polypeptide liquid; Polypeptide refining: perform membrane filtration, concentration, and drying on the lower polypeptide liquid to obtain alligator polypeptide products; Oil refining: perform deodorization, filtration, and gradient cooling on the upper crude oil to obtain refined alligator oil products; After the enzymatic hydrolysis and before the filtration, a composite additive is added to the enzymatic hydrolysate and stirred, and the composite additive comprises phospholipid, sucrose fatty acid ester with an HLB value of 13-16, and sucrose fatty acid ester with an HLB value of 5-9, and the mass ratio of the three is 1: (0.2-0.5) : (0.2-0.5); The addition of the composite additive adopts a step-by-step addition process, which specifically comprises: Add 40%-60% of the total amount of sucrose fatty acid ester with an HLB value of 13-16 to the enzymatic hydrolysate, and stir at a speed of 300-400 rpm for 5-8 minutes; Add a premix of phospholipid and all sucrose fatty acid ester with an HLB value of 5-9 to the enzymatic hydrolysate, and increase the stirring speed to 400-500 rpm for 15-20 minutes; Add the remaining sucrose fatty acid ester with an HLB value of 13-16, and adjust the stirring speed to 200-300 rpm for 5-10 minutes; the total action time is 34 minutes.

2. The preparation method for simultaneously and efficiently extracting crocodile polypeptides and crocodile oil as described in claim 1, characterized in that, In the filtration step, the adsorbent is white algae soil, and the addition amount is 3%-5% of the total mass of the enzymatic hydrolysate, and the temperature of the plate and frame filtration is 60℃.

3. The preparation method for simultaneously and efficiently extracting crocodile polypeptides and crocodile oil as described in claim 1, characterized in that, In the polypeptide refining step, the membrane filtration is sequentially performed through membrane systems with a pore size of 10,000 Da and 200 Da; in the oil refining step, the deodorization is ultrasonic deodorization under nitrogen protection, and the conditions are a frequency of 10-20 kHz, a power of 150 W, and a time of 30-60 s; the gradient cooling is from 60℃ to 25℃ at a rate of 5℃ / h.

4. The preparation method for simultaneously and efficiently extracting crocodile polypeptides and crocodile oil as described in claim 1, characterized in that, In the layering step and the oil refining step, the added amount of rosemary extract is 0.05%-0.1% of the total mass of the filtrate and 0.05%-0.1% of the total mass of the refined oil respectively.

5. The preparation method for simultaneously and efficiently extracting crocodile polypeptides and crocodile oil as described in claim 1, characterized in that, The phospholipid is food-grade soybean lecithin with purity ≥95% and particle size of 80-120 mesh; the sucrose fatty acid ester is food-grade with purity ≥98%.

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