A method for preparing an immunity-enhancing eel bone peptide composition

Abstract

This invention relates to a method for preparing an immune-enhancing eel bone peptide composition, belonging to the field of bioextraction technology. The method includes the following steps: a) Pretreatment: Eel bone material is sequentially freeze-dried at low temperature, pulverized to below 200 mesh, defatted with ethanol, and delimed using acid methods to obtain pretreated bone material; b) Three-stage complex enzymatic hydrolysis: The pretreated bone material is subjected to three-stage complex enzymatic hydrolysis to obtain an enzymatic hydrolysate; c) Enzyme inactivation and centrifugation: The enzymatic hydrolysate is inactivated and then centrifuged to remove insoluble matter, obtaining a clear liquid; d) Ultrafiltration separation: The clear liquid is subjected to ultrafiltration to remove macromolecular impurities, obtaining a collagen peptide concentrate; e) Debittering, deodorizing, and drying: The collagen peptide concentrate is debittered and deodorized, then dried to obtain eel bone collagen peptide powder. This method effectively removes fishy and bitter tastes, significantly improving the product's bioactivity, purity, and palatability.

Description

Technical Field

[0001] This invention belongs to the field of bio-extraction technology, specifically relating to a method for preparing an eel bone peptide composition that enhances immunity. Background Technology

[0002] Eel is a highly nutritious aquatic product, rich in high-quality protein, unsaturated fatty acids, and various trace elements. my country is the world's largest eel farming and processing country, processing approximately 200,000 tons of eel annually. During eel processing, a large amount of byproducts are generated, with eel bones accounting for about 15-20% of the fish's weight. These are mainly disposed of as waste or used as low-value feed, resulting in resource waste and environmental pollution.

[0003] Eel bones are rich in collagen, containing up to 30-40% of their dry weight. Enzymatic hydrolysis of collagen can produce peptides with various biological activities, including antioxidant, blood pressure-lowering, and immunomodulatory functions. In particular, collagen peptides with a molecular weight in the range of 3-10 kDa exhibit good bioavailability and immunomodulatory activity, showing broad application prospects in functional foods and health products.

[0004] However, existing methods for preparing fish bone collagen peptides have the following problems:

[0005] 1. Traditional enzymatic hydrolysis methods often use a single protease, resulting in low hydrolysis efficiency, unsatisfactory molecular weight distribution of the products, and low content of active peptides. Although some studies have used compound enzymatic hydrolysis, the selection and ratio of enzymes lack specificity, failing to fully leverage the synergistic effect.

[0006] 2. The minerals in fish bones (mainly hydroxyapatite) are tightly bound to collagen, forming a dense complex matrix that severely hinders the contact between proteases and substrates, leading to incomplete enzymatic hydrolysis. Existing methods do not provide precise control over the deashing process, resulting in either insufficient deashing leading to low enzymatic hydrolysis efficiency or excessive deashing damaging the collagen structure and affecting product quality.

[0007] 3. Traditional enzymatic hydrolysis methods are time-consuming and inefficient. Although some studies have attempted to use physical aids such as ultrasound and microwaves, these are mostly continuous ultrasound treatments, which can easily lead to excessive breakage of peptide chains and affect the bioactivity of the product.

[0008] 4. Fish protein peptide products generally suffer from a strong fishy smell and a noticeable bitter taste, which seriously affects consumer acceptance. Existing methods for removing fishy smell and bitterness have limited effectiveness and may significantly reduce the content of active ingredients in the products.

[0009] 5. Existing methods are not precise enough in controlling the molecular weight of products, often resulting in a large number of large protein molecules and small amino acids, and low purity of the target active peptide, which affects the functionality of the product.

[0010] Therefore, there is an urgent need to develop an efficient, environmentally friendly, and high-quality method for preparing eel bone collagen peptides in order to realize the high-value utilization of eel processing by-products. Summary of the Invention

[0011] The purpose of this invention is to provide a method for preparing an immune-enhancing eel bone peptide composition. This method uses an innovative three-stage compound enzymatic hydrolysis technology, combined with ultrasonic pulse treatment and precise debittering and deodorizing processes, to efficiently extract eel bone protein peptides with excellent immunomodulatory activity, while effectively removing fishy and bitter tastes, significantly improving the product's bioactivity, purity, and palatability, and meeting the high-quality demands of the functional food and health product market.

[0012] To achieve the above objectives, the technical solution adopted by the present invention is as follows:

[0013] a) Pretreatment: The eel bone material is successively subjected to low-temperature freeze drying, crushing to below 200 mesh, ethanol degreasing and acid deashing to obtain pretreated bone material;

[0014] b) Three-stage compound enzymatic hydrolysis: The pretreated aggregate is subjected to three-stage compound enzymatic hydrolysis to obtain the enzymatic hydrolysate;

[0015] c) Enzyme inactivation and centrifugation: After inactivating the enzyme in the enzyme hydrolysate, centrifuge to remove insoluble matter and obtain a clear liquid;

[0016] d) Ultrafiltration separation: The clear liquid is subjected to ultrafiltration separation to remove macromolecular impurities and obtain collagen peptide concentrate;

[0017] e) Debittering and deodorizing, and drying: The collagen peptide concentrate is debittered and deodorized, and then dried to obtain eel bone collagen peptide powder.

[0018] Specifically, the conditions for ethanol degreasing in step a) are as follows: use 95% (v / v) ethanol, degreasing temperature 4-8℃, process twice, each time for 2-3 hours, with a material-to-liquid mass ratio of 1:(3-5); after degreasing, vacuum dry at 40-50℃ to constant weight to ensure that the residual ethanol content is ≤500 mg / kg.

[0019] Specifically, the conditions for acid deashing are as follows: The defatted aggregate is treated with a 0.3-0.5 mol / L lactic acid solution at a mass ratio of 1:(8-10), and stirred at 4-8℃ for 2-4 hours. During deashing, samples are taken every 30 minutes to check the ash content. Deashing is stopped when the ash content drops to 6.5-8.0%, at which point the aggregate is translucent and soft. The aggregate is repeatedly rinsed with deionized water until neutral (pH 6.5-7.0). After each rinse, the calcium ion concentration of the rinsing solution is checked; rinsing is considered complete when the calcium ion concentration is <50 mg / L. The ash content of the pretreated aggregate after dehydration should be controlled between 6.5-8.0%.

[0020] The control of ash content has a significant impact on the subsequent enzymatic hydrolysis effect: when the ash content is >8%, residual mineral crystals will coat collagen fibers, hindering the contact between proteases and substrates, resulting in a 10-15% decrease in enzymatic hydrolysis efficiency and a reduction in the final product yield; when the ash content is <6.5%, although the enzymatic hydrolysis efficiency is improved, excessive deashing will damage part of the collagen fiber structure, leading to the production of too many small molecule peptides (<3kDa), affecting the molecular weight distribution and immunomodulatory activity of the product. Therefore, controlling the ash content between 6.5% and 8.0% can ensure enzymatic hydrolysis efficiency while obtaining the optimal molecular weight distribution and bioactivity.

[0021] Specifically, the parameters for the three-stage complex enzymatic hydrolysis described in step b) are as follows: First stage: add neutral protease, adjust pH to 7.0, temperature 50-55℃, and hydrolyze for 2 hours; Second stage: add Asp-N protease, adjust pH to 7.5-8.5, temperature 40-45℃, and hydrolyze for 4-6 hours; Third stage: add flavor protease, adjust pH to 6.5-7.0, temperature 45-50℃, and hydrolyze for 1-2 hours; The material-to-liquid mass ratio is maintained at 1:(8-12) during each stage of enzymatic hydrolysis.

[0022] Specifically, in step b), the second stage of enzymatic hydrolysis is combined with ultrasonic pulse treatment. Ultrasonic treatment is applied throughout the entire second stage of enzymatic hydrolysis: the first half uses a frequency of 40kHz, 350W, with a 3s operation followed by a 1s pause; the second half uses a frequency of 20kHz, 280W, with a 2s operation followed by a 4s pause. The mechanical vibration and cavitation effect of ultrasound can disrupt the collagen fiber bundle structure and the collagen-hydroxyapatite complex matrix, enhancing the effective contact between the enzyme and the collagen substrate, promoting enzyme-substrate contact. Simultaneously, appropriate frequency and power settings can avoid excessive damage to the peptide chains, significantly improving enzymatic hydrolysis efficiency and the bioactivity of the product.

[0023] Specifically, in the compound enzyme, the mass ratio of neutral protease, Asp-N protease and flavor protease is (1-1.7):(2.7-4):(0.7-1.3), and the total amount of compound enzyme added is 1.3-1.9% of the mass of pretreated aggregate.

[0024] Specifically, the enzyme inactivation conditions in step c) are a temperature of 95°C and a time of 10 min; the centrifugation conditions are a temperature of 4°C, a rotation speed of 4000-6000 rpm, and a time of 15-20 min.

[0025] Specifically, in step d), ultrafiltration separation is carried out in two stages: the first stage uses an ultrafiltration membrane with a molecular weight cutoff of 10 kDa, operating at a pressure of 0.15-0.25 MPa, and collects the permeate to remove large molecular weight proteins; the second stage uses an ultrafiltration membrane with a molecular weight cutoff of 3 kDa, operating at a pressure of 0.10-0.20 MPa, collects the retentate and concentrates it 3-5 times to obtain a collagen peptide concentrate with a molecular weight of 3-10 kDa.

[0026] Specifically, in step e), the collagen peptide concentrate is cooled to 35-40℃, and debittering exopeptidase with an enzyme activity of 50-100 U / mg is added at a concentration of 0.1-0.3% of the concentrate mass. After treatment for 30 min, the enzyme is inactivated at 95℃ for 10 min, and then cooled to room temperature. Activated carbon of 1-2% of the concentrate mass is added and stirred for adsorption for 30 min, while nitrogen is purged at a flow rate of 200-300 mL / min for 30 min. After filtration to remove the activated carbon, the solution is dried.

[0027] The drying process can be achieved using either spray drying or freeze drying.

[0028] Spray drying is suitable for large-scale industrial production, offering fast drying speed and low cost. The concentrated liquid, after debittering and deodorizing treatment, is fed into the spray drying tower via a high-pressure pump (1.5-2.5 MPa). The feed rate is 30-40 L / h, the hot air inlet temperature is 120-140℃, the outlet temperature is 60-80℃, and the atomizer speed is 18000-22000 rpm. The product has a residual moisture content of ≤6%, a particle size of 50-150 μm, a bulk density of 0.35-0.45 g / cm³, and exhibits good flowability and rapid solubility. Spray drying is suitable for applications requiring high product appearance and rapid solubility.

[0029] Freeze-drying: Suitable for small-batch production or applications requiring extremely high product activity, it maximizes the preservation of the product's bioactivity and nutritional components. The concentrated liquid, after debittering and deodorizing treatment, is pre-frozen to -40℃ for 2-4 hours, then subjected to a first drying process (-20℃ to -25℃, 8-12 hours, vacuum 5-15 Pa) and a second desorption drying process (20-30℃, 4-8 hours, vacuum 3-8 Pa) in a freeze dryer. The product has a residual moisture content of ≤4%, a loose porous structure, irregular particles, a bulk density of 0.15-0.25 g / cm³, and excellent rehydration properties. The immunomodulatory activity of freeze-dried products is 5-8% higher than that of spray-dried products, but the production cost is approximately 3-5 times that of spray-dried products.

[0030] The products obtained by the two drying methods have no significant differences in protein content, molecular weight distribution and basic physicochemical properties. The appropriate drying method can be selected according to the production scale, cost requirements and application scenarios.

[0031] Specifically, the eel bone material is a by-product of processing fresh eel, with a water content controlled below 10% and a protein content ≥15%.

[0032] The beneficial effects of this invention are as follows:

[0033] 1. The application of a three-stage complex enzymatic hydrolysis technology significantly improves the hydrolysis rate of proteins and the bioactivity of peptides. Through the synergistic effect of different enzymes, collagen in eel bones can be hydrolyzed more comprehensively, producing peptides with specific bioactivities. In particular, the use of Asp-N protease can produce aspartic acid-rich active peptides, which have unique advantages in immune regulation.

[0034] 2. The introduction of ultrasonic pulse processing technology significantly improves enzymatic hydrolysis efficiency. The mechanical and cavitation effects of ultrasound can disrupt the collagen fiber bundle structure and the collagen-hydroxyapatite complex matrix, increasing the contact area between the enzyme and the substrate, while promoting mass transfer and making the enzymatic hydrolysis reaction more complete. Compared with traditional enzymatic hydrolysis methods, the method of this invention can achieve higher protein recovery rates and better peptide distribution in a shorter time.

[0035] 3. Precise molecular weight control ensures the product's bioactivity and functionality. Through two-stage ultrafiltration separation, the molecular weight distribution of peptides in the product can be precisely controlled, removing inactive large-molecule proteins and small-molecule amino acids, and enriching medium-molecular-weight peptides with immunomodulatory activity, significantly improving the product's functionality.

[0036] 4. The innovative debittering and deodorizing process significantly improves the product's palatability. Traditional fish protein peptide products often suffer from a strong fishy smell and a noticeable bitterness, severely impacting consumer acceptance. This invention, through debittering exopeptidase treatment combined with activated carbon adsorption and nitrogen replacement, effectively removes bitterness and fishy smell, resulting in a product with excellent taste and flavor.

[0037] 5. The entire preparation process is simple, easy to operate, and cost-controllable. The equipment and technology used are mature industrial technologies, which are easy to implement for large-scale production. At the same time, the raw materials are derived from by-products of eel processing, turning waste into treasure and having good economic and environmental benefits.

[0038] 6. The prepared eel bone collagen peptides possess excellent immunomodulatory activity. The product is rich in immunomodulatory active peptides, which can effectively regulate the body's immune function, enhance disease resistance, and are suitable for the development of functional foods and health products. Detailed Implementation

[0039] The present invention will be further described below with reference to specific embodiments, but the invention is not limited to these embodiments. Those skilled in the art should recognize that the present invention covers all possible alternatives, improvements, and equivalents included within the scope of the claims.

[0040] Unless otherwise stated, pH adjustment in all embodiments of this invention uses the following reagents and methods:

[0041] When it is necessary to increase the pH value, use a 1 mol / L sodium hydroxide solution, and control the dropping rate at 2-5 mL / min. At the same time, monitor the pH value in real time with a pH meter, and stop dropping after the target pH value is reached.

[0042] When it is necessary to lower the pH value, use a 1 mol / L hydrochloric acid solution, and control the dropping rate at 2-5 mL / min. At the same time, monitor the pH value in real time with a pH meter, and stop dropping after the target pH value is reached.

[0043] During pH adjustment, the solution should be kept thoroughly stirred (300-500 rpm) to ensure a uniform pH distribution;

[0044] All pH measurements were performed at the reaction temperature using pH meters calibrated with standard buffer solutions (pH 4.00, 7.00, 10.00).

[0045] Neutral protease (purchased from Novozymes, catalog number Neutrase 0.8L, activity ≥0.8 AU / g), Asp-N protease (purchased from Sigma-Aldrich, catalog number P3303, activity ≥150 U / g), and flavor protease (purchased from Novozymes, catalog number Flavorzyme 1000L, activity ≥1000 LAPU / g).

[0046] The following is the drying process of the example: Spray drying: feed rate 30-40L / h, hot air inlet temperature 120-140℃, outlet temperature 60-80℃, moisture residue ≤6%; Freeze drying: pre-freezing temperature -40℃ (2-4h), primary drying -20℃ to -25℃ (8-12h, vacuum degree 5-15Pa), desorption drying 20-30℃ (4-8h, vacuum degree 3-8Pa), final moisture ≤4%.

[0047] Example 1;

[0048] a) Preprocessing:

[0049] Take 1200g of fresh eel bone material (8-9% water content, 16-17% protein content), rinse it with tap water to remove surface blood and residual tissue, drain it and place it in a freeze dryer at -40℃ for 24 hours until the water content is <5%.

[0050] The freeze-dried aggregate was fed into a high-speed pulverizer and pulverized to below 200 mesh (particle size <74μm) to obtain approximately 900g of bone meal.

[0051] Ethanol degreasing was performed at a degreasing temperature of 6℃ and a material-to-liquid mass ratio of 1:4: 900g of bone meal was mixed with 3600mL of 95% ethanol solution and placed in a refrigerated environment at 4-8℃. The mixture was mechanically stirred (200 rpm) for 3 hours. After filtration, the ethanol was discarded. The bone meal was then degreased a second time with fresh 95% ethanol solution (3600mL) under the same conditions for 3 hours. The degreased bone meal was dried in a vacuum drying oven at 45℃ until constant weight (approximately 6 hours). The residual ethanol content was measured to be 420 mg / kg, which met the requirements.

[0052] Acid deashing: 850g of defatted aggregate was mixed with 7650mL of 0.4 mol / L lactic acid solution (aggregate-liquid mass ratio 1:9) and placed in an environment of 4-8℃. Deashing was carried out by mechanical stirring (150 rpm). Approximately 2g samples were taken every 30 minutes, and the ash content was determined using the muffle furnace ashing method (550℃, 4h). After approximately 3 hours of deashing, the ash content decreased to 7.2%, and the aggregate became translucent and soft; deashing was then stopped.

[0053] Rinse the deashed aggregate repeatedly with deionized water, using approximately 5L of water each time. Gently stir for 10 minutes, then allow to settle and discard the supernatant. Repeat the rinsing process 6 times until the pH of the rinsing solution reaches 6.8. Take the last rinsing solution and test the calcium ion concentration; if it is 38 mg / L (<50 mg / L standard), the rinsing is considered complete.

[0054] The rinsed aggregate was wrapped in gauze and squeezed to remove water, and then dried in a vacuum drying oven at 45°C to constant weight (about 8 hours) to obtain 720g of pretreated aggregate, which is a white to light yellow powder with an ash content of 7.2% and a protein content (on a dry basis) of 68.5%.

[0055] b) Three-stage complex enzymatic hydrolysis:

[0056] Add 720g of pretreated aggregate to a 10L stainless steel enzymatic hydrolysis reactor, add 7200mL of deionized water at a mass ratio of 1:10, and stir to mix evenly.

[0057] Preparation of compound enzymes: Weigh 1.50g of neutral protease (activity 0.8 AU / g), 3.80g of Asp-N protease (activity 150 U / g), and 1.15g of flavor protease (activity 1000 LAPU / g). The total amount of compound enzymes added is 1.6% (11.52g) of the mass of the pretreated aggregate.

[0058] First stage of enzymatic hydrolysis: Heat the reactor to 52℃, adjust the pH to 7.0 with 1 mol / L sodium hydroxide solution (approximately 15 mL), add 1.50 g of neutral protease, and mechanically stir at 300 rpm for 2 hours. During the hydrolysis process, monitor the pH every 30 minutes. If the pH drops by more than 0.2 units, add a small amount of 1 mol / L sodium hydroxide solution to maintain the pH at 7.0 ± 0.1. Control the temperature at 52 ± 1℃.

[0059] Second stage of enzymatic hydrolysis: Add 3.80 g of Asp-N protease to the reaction vessel, adjust the pH to 8.0 with 1 mol / L sodium hydroxide solution (approximately 25 mL), adjust the temperature to 42℃, and hydrolyze for 5 hours. Simultaneously, perform ultrasonic pulse treatment.

[0060] Using a dual-frequency ultrasonic generator (model JY92-IIN, Ningbo Xinzhi Biotechnology Co., Ltd.), the ultrasonic probe (titanium alloy, 28mm diameter) was vertically inserted into the reactor to a depth of 10cm below the liquid surface (the liquid level in the reactor was approximately 15cm, and the probe was approximately 5cm from the bottom of the reactor). The circulating water cooling system was started, with the cooling water temperature set to 18℃ and the flow rate to 600 mL / min.

[0061] The first 2.5 hours: The ultrasonic generator was set to a frequency of 40kHz, a power of 350W, and a pulse mode of 3 seconds of operation followed by a 1-second interval. The actual working time accounted for 75%, with an effective ultrasonic processing time of 1.875 hours.

[0062] In the last 2.5 hours: the ultrasonic generator was switched to a frequency of 20kHz, a power of 280W, and a pulse mode of 2 seconds of operation followed by a 4-second interval. The actual working time accounted for 33.3%, with an effective ultrasonic processing time of 0.833 hours.

[0063] During ultrasonic treatment, mechanical stirring was reduced to 250 rpm, and the temperature of the reaction solution was monitored every 30 minutes, maintaining the temperature range at 41-43℃ and the pH value at 8.0±0.1 (if necessary, 1 mol / L sodium hydroxide solution was added, for a total of about 10 mL).

[0064] The third stage of enzymatic hydrolysis: Add 1.15g of flavor protease to the reactor, adjust the pH to 6.8 with 1mol / L hydrochloric acid solution (approximately 30mL), adjust the temperature to 47℃, stop ultrasonic treatment, and restore mechanical stirring to 300 rpm. Hydrolyze for 1.5h. During the enzymatic hydrolysis process, maintain the pH at 6.8±0.1 and control the temperature at 47±1℃.

[0065] The three-stage enzymatic hydrolysis process took a total of 8.5 hours, yielding approximately 8000 mL of hydrolysate, which was a uniform brownish-yellow liquid with a characteristic enzymatic odor.

[0066] c) Enzyme inactivation and centrifugation:

[0067] After enzymatic hydrolysis, the reaction vessel was rapidly heated to 95°C and held for 10 minutes to inactivate the enzyme, then quickly cooled to room temperature. The hydrolysate was transferred to a refrigerator and allowed to stand at 4°C for 2 hours to allow the insoluble matter to settle completely.

[0068] The enzymatic hydrolysate was poured into a high-speed centrifuge in batches (1000 mL per batch). The centrifugation conditions were set as follows: temperature 4℃, speed 5000 rpm, and centrifugation time 18 min. After centrifugation, the supernatant was carefully aspirated, and the precipitate (mainly incompletely hydrolyzed bone residue and denatured protein precipitate, accounting for about 35% of the total volume) was discarded.

[0069] Combine all the supernatants from centrifugation to obtain approximately 5200 mL of clear liquid, which is a light brownish-yellow clear liquid with a transmittance >85%.

[0070] d) Ultrafiltration separation:

[0071] The clarified liquid is then processed through a two-stage ultrafiltration system:

[0072] First-stage ultrafiltration: A polyethersulfone ultrafiltration membrane with a molecular weight cutoff of 10 kDa (effective membrane area 0.8 m²) is used. 2 The operating pressure is set to 0.20 MPa, and the temperature is controlled at 15-20℃. The supernatant is passed through the ultrafiltration membrane at a flow rate of approximately 150 mL / min, and the permeate is collected to remove incompletely enzymatically hydrolyzed large protein molecules (molecular weight > 10 kDa). The retentate (approximately 800 mL, containing large protein molecules) is discarded, and approximately 4400 mL of permeate enters the second-stage ultrafiltration.

[0073] Second-stage ultrafiltration: A polyethersulfone ultrafiltration membrane with a molecular weight cutoff of 3 kDa (effective membrane area 0.8 m²) is used. 2 The operating pressure is set to 0.15 MPa, and the temperature is controlled at 15-20℃. The first-stage permeate is passed through the ultrafiltration membrane at a flow rate of approximately 120 mL / min. The permeate (approximately 3200 mL, mainly consisting of small molecule amino acids and peptides with a molecular weight <3 kDa) is discarded, and the retentate is collected.

[0074] Continue ultrafiltration to concentrate the retentate. Stop ultrafiltration when the volume is concentrated to about 1200 mL (the concentration factor is about 4 times, relative to the volume of permeate before the second stage of ultrafiltration). 1200 mL of collagen peptide concentrate with a molecular weight of 3-10 kDa is obtained. It is a brownish-yellow viscous liquid with a solid content of about 15%.

[0075] e) Removing bitterness and fishy smell, and drying:

[0076] Transfer 1200 mL of collagen peptide concentrate to a reaction vessel and cool to 37°C. Add 2.4 g of debittering exopeptidase (enzyme activity 75 U / mg) (0.2% of the concentrate mass, which is approximately 1260 g based on a density of 1.05 g / mL). Gently stir (100 rpm) for 30 min. After debittering, rapidly heat the reaction solution to 95°C and hold for 10 min to inactivate the enzyme, then cool to room temperature.

[0077] Add 18.9 g of activated carbon (1.5% of the concentrate mass) to the cooled concentrate and stir for 30 min (150 rpm) for adsorption. During the adsorption process, simultaneously introduce high-purity nitrogen gas (99.99% purity) at a flow rate of 250 mL / min, evenly dispersed in the liquid through an aeration head, and perform nitrogen replacement for 30 min to remove dissolved oxygen and volatile fishy-smelling substances.

[0078] After adsorption and nitrogen replacement, the activated carbon is removed by filtration with multiple layers of gauze and filter paper, resulting in a concentrated solution of about 1150 mL after debittering and deodorizing treatment. The solution is a light yellow, clear liquid with a characteristic slight fishy aroma and no obvious bitterness or fishy smell.

[0079] Drying process: Considering that this embodiment is used to evaluate the maximum immunogenic potential of the product, freeze drying is used to preserve the bioactivity of the active peptides to the maximum extent.

[0080] After debittering and deodorizing, the concentrated liquid was dispensed into freeze-drying trays (approximately 200 mL per tray, with a thickness of approximately 1 cm) and placed in a -40°C freezer for 3 hours to allow the concentrated liquid to freeze completely into a solid.

[0081] Transfer the frozen sample to a freeze dryer (FD-1A-50 model) and freeze dry it according to the following procedure:

[0082] First drying: -22℃, 10h, vacuum degree 8Pa, sublimation to remove ice crystals.

[0083] Drying: Slowly raise the temperature to 22°C and maintain for 5 hours under a vacuum of 4 Pa ​​to remove residual moisture.

[0084] After freeze-drying, 185g of eel bone collagen peptide powder was obtained. It was a loose, porous powder, ranging from white to pale yellow, with irregular particles and a bulk density of about 0.20 g / cm³.

[0085] Example 2;

[0086] a) Preprocessing:

[0087] Take 1000g of fresh eel bone material (moisture content 9-10%, protein content 15-16%), wash it with clean water to remove blood and impurities from the surface, drain it and place it in a freeze dryer, freeze dry at -40℃ for 24 hours until the moisture content is <5%.

[0088] The freeze-dried aggregate was fed into a high-speed pulverizer and pulverized to below 200 mesh (particle size <74μm) to obtain approximately 750g of bone meal.

[0089] Ethanol degreasing was performed at a degreasing temperature of 4℃ and a material-to-liquid mass ratio of 1:3: 750g of bone meal was mixed with 2250mL of 95% ethanol solution and placed in a refrigerated environment at 4℃. The mixture was mechanically stirred (200 rpm) for 2 hours. After filtration, the ethanol was discarded. The bone meal was then degreased a second time with 2250mL of fresh 95% ethanol solution under the same conditions for 2 hours. The degreased bone meal was dried in a vacuum drying oven at 40℃ until constant weight (approximately 5 hours). The residual ethanol content was measured to be 380 mg / kg, which met the requirements.

[0090] Acid deashing: 700g of defatted aggregate was mixed with 5600mL of 0.3 mol / L lactic acid solution (aggregate-liquid mass ratio 1:8) and placed in an environment of 4℃. Deashing was carried out by mechanical stirring (150 rpm). Approximately 2g samples were taken every 30 minutes, and the ash content was determined using the muffle furnace ashing method (550℃, 4h). After approximately 2 hours of deashing, the ash content decreased to 7.8%, and the aggregate became translucent and soft; deashing was then stopped.

[0091] Rinse the deashed aggregate repeatedly with deionized water, using approximately 4L of water each time. Gently stir for 10 minutes, then allow to settle and discard the supernatant. Repeat the rinsing process 7 times until the pH of the rinsing solution reaches 7.0. Take the last rinsing solution and test the calcium ion concentration; if it is 42 mg / L (<50 mg / L standard), the rinsing is considered complete.

[0092] The rinsed aggregate was wrapped in gauze and squeezed to remove water, and then dried in a vacuum drying oven at 40°C to constant weight (about 7 hours) to obtain 580g of pretreated aggregate, which is a white to light yellow powder with an ash content of 7.8% and a protein content (on a dry basis) of 66.8%.

[0093] b) Three-stage complex enzymatic hydrolysis:

[0094] Add 580g of pretreated aggregate to an 8L stainless steel enzymatic hydrolysis reactor, add 4640mL of deionized water at a mass ratio of 1:8, and stir to mix evenly.

[0095] Preparation of compound enzymes: Weigh 0.75g of neutral protease (activity 0.8 AU / g), 2.03g of Asp-N protease (activity 150 U / g), and 0.53g of flavor protease (activity 1000 LAPU / g). The total amount of compound enzymes added is 1.3% (7.54g) of the mass of the pretreated aggregate.

[0096] First stage of enzymatic hydrolysis: Heat the reactor to 50℃, adjust the pH to 7.0 with 1 mol / L sodium hydroxide solution (approximately 12 mL), add 0.75 g of neutral protease, and mechanically stir at 300 rpm for 2 hours. During the hydrolysis process, monitor the pH every 30 minutes. If the pH drops by more than 0.2 units, add a small amount of 1 mol / L sodium hydroxide solution to maintain the pH at 7.0 ± 0.1. Control the temperature at 50 ± 1℃.

[0097] Second stage of enzymatic hydrolysis: Add 2.03g of Asp-N protease to the reaction vessel, adjust the pH to 7.5 with 1 mol / L sodium hydroxide solution (approximately 18mL required), adjust the temperature to 40℃, and hydrolyze for 4 hours. Simultaneously, perform ultrasonic pulse treatment.

[0098] Using a dual-frequency ultrasonic generator (model JY92-IIN), vertically insert the ultrasonic probe (titanium alloy, 26mm diameter) into the reactor to a depth of 9cm below the liquid surface (the liquid level in the reactor is approximately 13cm, and the probe is approximately 4cm from the bottom of the reactor). Start the circulating water cooling system, setting the cooling water temperature to 16℃ and the flow rate to 550 mL / min.

[0099] The first 2 hours: The ultrasonic generator was set to a frequency of 40kHz, a power of 350W, and a pulse mode of 3 seconds of operation followed by a 1-second interval. The actual working time accounted for 75%, with an effective ultrasonic processing time of 1.5 hours.

[0100] In the last 2 hours: the ultrasonic generator was switched to a frequency of 20kHz, a power of 280W, and a pulse mode of 2 seconds of operation followed by a 4-second interval. The actual working time accounted for 33.3%, with an effective ultrasonic processing time of 0.667 hours.

[0101] During ultrasonic treatment, mechanical stirring was reduced to 250 rpm, and the temperature of the reaction solution was monitored every 30 minutes, maintaining the temperature range at 39-41℃ and the pH value at 7.5±0.1 (if necessary, 1 mol / L sodium hydroxide solution was added, for a total of about 8 mL).

[0102] The third stage of enzymatic hydrolysis: Add 0.53g of flavor protease to the reactor, adjust the pH to 6.5 with 1 mol / L hydrochloric acid solution (approximately 22mL required), adjust the temperature to 45℃, stop ultrasonic treatment, and restore mechanical stirring to 300 rpm. Hydrolyze for 1 hour. During the enzymatic hydrolysis process, maintain the pH at 6.5±0.1 and control the temperature at 45±1℃.

[0103] The three-stage enzymatic hydrolysis process took a total of 7 hours, yielding approximately 5200 mL of hydrolysate, which was a uniform brownish-yellow liquid with a characteristic enzymatic odor.

[0104] c) Enzyme inactivation and centrifugation:

[0105] After enzymatic hydrolysis, the reaction vessel was rapidly heated to 95°C and held for 10 minutes to inactivate the enzyme, then quickly cooled to room temperature. The hydrolysate was transferred to a refrigerator and allowed to stand at 4°C for 2 hours to allow the insoluble matter to settle completely.

[0106] The enzymatic hydrolysate was poured into a high-speed centrifuge in batches (1000 mL each). The centrifugation conditions were set as follows: temperature 4℃, speed 4000 rpm, and centrifugation time 15 min. After centrifugation, the supernatant was carefully aspirated, and the precipitate (mainly incompletely hydrolyzed bone fragments and denatured protein precipitates, accounting for approximately 27% of the total volume) was discarded.

[0107] Combine all the supernatants from centrifugation to obtain approximately 3800 mL of clear liquid, which is a light brownish-yellow clear liquid with a transmittance of >80%.

[0108] d) Ultrafiltration separation:

[0109] The clarified liquid is then processed through a two-stage ultrafiltration system:

[0110] First-stage ultrafiltration: A polyethersulfone ultrafiltration membrane with a molecular weight cutoff of 10 kDa (effective membrane area 0.6 m²) is used. 2 The operating pressure is set to 0.15 MPa, and the temperature is controlled at 15-20℃. The supernatant is passed through the ultrafiltration membrane at a flow rate of approximately 140 mL / min, and the permeate is collected to remove incompletely enzymatically hydrolyzed large protein molecules (molecular weight > 10 kDa). The retentate (approximately 600 mL, containing large protein molecules) is discarded, and approximately 3200 mL of permeate enters the second-stage ultrafiltration.

[0111] Second-stage ultrafiltration: A polyethersulfone ultrafiltration membrane with a molecular weight cutoff of 3 kDa (effective membrane area 0.6 m²) is used. 2 The operating pressure is set to 0.10 MPa, and the temperature is controlled at 15-20℃. The first-stage permeate is passed through the ultrafiltration membrane at a flow rate of approximately 110 mL / min. The permeate (approximately 2300 mL, mainly consisting of small molecule amino acids and peptides with a molecular weight <3 kDa) is discarded, and the retentate is collected.

[0112] Continue ultrafiltration to concentrate the retentate. Stop ultrafiltration when the volume is concentrated to about 900 mL (the concentration factor is about 3 times, relative to the volume of permeate before the second stage of ultrafiltration). 900 mL of collagen peptide concentrate with a molecular weight of 3-10 kDa is obtained. It is a brownish-yellow viscous liquid with a solid content of about 16%.

[0113] e) Removing bitterness and fishy smell, and drying:

[0114] Transfer 900 mL of collagen peptide concentrate to a reaction vessel and cool to 35°C. Add 0.95 g of debittering exopeptidase (enzyme activity 50 U / mg) (0.1% of the concentrate mass, which is approximately 945 g based on a density of 1.05 g / mL). Gently stir (100 rpm) for 30 min. After debittering, rapidly heat the reaction solution to 95°C and hold for 10 min to inactivate the enzyme, then cool to room temperature.

[0115] Add 9.45 g of activated carbon (1.0% of the concentrate mass) to the cooled concentrate and stir for 30 min (150 rpm) for adsorption. During the adsorption process, simultaneously introduce high-purity nitrogen gas (99.99% purity) at a flow rate of 200 mL / min, evenly dispersed in the liquid through an aeration head, and perform nitrogen replacement for 30 min to remove dissolved oxygen and volatile fishy-smelling substances.

[0116] After adsorption and nitrogen replacement, the activated carbon is removed by filtration with multiple layers of gauze and filter paper, resulting in a concentrated solution of about 870 mL after debittering and deodorizing treatment. The solution is a light yellow, clear liquid with a characteristic slight fishy aroma and no obvious bitterness or fishy smell.

[0117] Drying process: Considering the cost-effectiveness of industrial production, spray drying is adopted.

[0118] 870 mL of the concentrated solution after debittering and deodorizing treatment was sent to a spray dryer (LPG-5 type) for spray drying. The operating parameters were set as follows:

[0119] Feed rate: 35L / h (the concentrate is diluted to the appropriate concentration).

[0120] Inlet air temperature: 120℃

[0121] Air outlet temperature: 60℃

[0122] Atomizer speed: 20000 rpm

[0123] Atomization pressure: 2.0 MPa

[0124] The spray drying process takes about 25 minutes, yielding 145g of eel bone collagen peptide powder. The powder is a light yellow fine powder with a particle size of 80-120μm, a bulk density of about 0.40 g / cm³, and good flowability.

[0125] Example 3;

[0126] a) Preprocessing:

[0127] Take 800g of fresh eel bone material (7-8% water content, 17-18% protein content), wash it with clean water to remove surface blood and residual tissue, drain it and place it in a freeze dryer, freeze dry at -40℃ for 24 hours until the water content is <5%.

[0128] The freeze-dried aggregate was fed into a high-speed pulverizer and pulverized to below 200 mesh (particle size <74μm) to obtain approximately 620g of bone meal.

[0129] Ethanol degreasing was performed at a degreasing temperature of 8℃ and a material-to-liquid mass ratio of 1:5: 620g of bone meal was mixed with 3100mL of 95% ethanol solution and placed in a refrigerated environment at 4-8℃. The mixture was mechanically stirred (200 rpm) for 4 hours. After filtration, the ethanol was discarded. The bone meal was then degreased a second time with fresh 95% ethanol solution (3100mL) under the same conditions for 4 hours. The degreased bone meal was dried in a vacuum drying oven at 50℃ until constant weight (approximately 7 hours). The residual ethanol content was measured to be 460 mg / kg, which met the requirements.

[0130] Acid deashing: 580g of defatted aggregate was mixed with 5800mL of 0.5 mol / L lactic acid solution (aggregate-liquid mass ratio 1:10) and placed in an 8℃ environment. Deashing was performed by mechanical stirring (150 rpm). Approximately 2g samples were taken every 30 minutes, and the ash content was determined using a muffle furnace ashing method (550℃, 4h). After approximately 4 hours of deashing, the ash content decreased to 6.8%, and the aggregate became translucent and soft; deashing was then stopped.

[0131] Rinse the deashed aggregate repeatedly with deionized water, using approximately 5L of water each time. Gently stir for 10 minutes, then allow to settle and discard the supernatant. Repeat the rinsing process 8 times until the pH of the rinsing solution reaches 6.5. Take the last rinsing solution and test the calcium ion concentration; if it is 45 mg / L (<50 mg / L standard), the rinsing is considered complete.

[0132] The rinsed aggregate was wrapped in gauze and squeezed to remove water, and then dried in a vacuum drying oven at 50°C to constant weight (about 9 hours) to obtain 480g of pretreated aggregate, which is a white to light yellow powder with an ash content of 6.8% and a protein content (on a dry basis) of 70.2%.

[0133] b) Three-stage complex enzymatic hydrolysis:

[0134] Add 480g of pretreated aggregate to an 8L stainless steel enzymatic hydrolysis reactor, add 5760mL of deionized water at a mass ratio of 1:12, and stir to mix evenly.

[0135] Preparation of compound enzymes: Weigh 1.55g of neutral protease (activity 0.8 AU / g), 3.65g of Asp-N protease (activity 150 U / g), and 1.18g of flavor protease (activity 1000 LAPU / g). The total amount of compound enzymes added is 1.9% (9.12g) of the mass of the pretreated aggregate.

[0136] First stage of enzymatic hydrolysis: Heat the reactor to 55℃, adjust the pH to 7.0 with 1 mol / L sodium hydroxide solution (approximately 14 mL), add 1.55 g of neutral protease, and mechanically stir at 300 rpm for 2 hours. During the hydrolysis process, monitor the pH every 30 minutes. If the pH drops by more than 0.2 units, add a small amount of 1 mol / L sodium hydroxide solution to maintain the pH at 7.0 ± 0.1. Control the temperature at 55 ± 1℃.

[0137] Second stage of enzymatic hydrolysis: Add 3.65g of Asp-N protease to the reaction vessel, adjust the pH to 8.5 with 1 mol / L sodium hydroxide solution (approximately 28mL required), adjust the temperature to 45℃, and hydrolyze for 6 hours. Simultaneously, perform ultrasonic pulse treatment.

[0138] Using a dual-frequency ultrasonic generator (model JY92-IIN), vertically insert the ultrasonic probe (titanium alloy, 30mm diameter) into the reactor to a depth of 11cm below the liquid surface (the liquid level in the reactor is approximately 17cm, and the probe is approximately 6cm from the bottom of the reactor). Start the circulating water cooling system, setting the cooling water temperature to 20℃ and the flow rate to 700 mL / min.

[0139] The first 3 hours: The ultrasonic generator was set to a frequency of 40kHz, a power of 350W, and a pulse mode of 3 seconds of operation followed by a 1-second interval. The actual working time accounted for 75%, with an effective ultrasonic processing time of 2.25 hours.

[0140] In the last 3 hours: the ultrasonic generator was switched to a frequency of 20kHz, a power of 280W, and a pulse mode of 2 seconds of operation followed by a 4-second interval. The actual working time accounted for 33.3%, with an effective ultrasonic processing time of 1.0 hour.

[0141] During ultrasonic treatment, mechanical stirring was reduced to 250 rpm, and the temperature of the reaction solution was monitored every 30 minutes, maintaining the temperature range at 43-45℃ and the pH value at 8.5±0.1 (if necessary, 1 mol / L sodium hydroxide solution was added, for a total of about 12 mL).

[0142] The third stage of enzymatic hydrolysis: Add 1.18g of flavor protease to the reactor, adjust the pH to 7.0 with 1mol / L hydrochloric acid solution (approximately 35mL required), adjust the temperature to 50℃, stop the ultrasonic treatment, and restore the mechanical stirring to 300 rpm. Hydrolyze for 2 hours. During the enzymatic hydrolysis process, maintain the pH at 7.0±0.1 and control the temperature at 50±1℃.

[0143] The three-stage enzymatic hydrolysis process took a total of 10 hours, yielding approximately 6500 mL of hydrolysate, which was a uniform brownish-yellow liquid with a characteristic enzymatic odor.

[0144] c) Enzyme inactivation and centrifugation:

[0145] After enzymatic hydrolysis, the reaction vessel was rapidly heated to 95°C and held for 10 minutes to inactivate the enzyme, then quickly cooled to room temperature. The hydrolysate was transferred to a refrigerator and allowed to stand at 4°C for 2 hours to allow the insoluble matter to settle completely.

[0146] The enzymatic hydrolysate was poured into a high-speed centrifuge in batches (1000 mL per batch). The centrifugation conditions were set as follows: temperature 4℃, speed 6000 rpm, and centrifugation time 20 min. After centrifugation, the supernatant was carefully aspirated, and the precipitate (mainly incompletely hydrolyzed bone fragments and denatured protein precipitates, accounting for approximately 26% of the total volume) was discarded.

[0147] Combine all the supernatants from centrifugation to obtain approximately 4800 mL of clear liquid, which is a light brownish-yellow clear liquid with a transmittance of >88%.

[0148] d) Ultrafiltration separation:

[0149] The clarified liquid is then processed through a two-stage ultrafiltration system:

[0150] First-stage ultrafiltration: A polyethersulfone ultrafiltration membrane with a molecular weight cutoff of 10 kDa (effective membrane area 1.0 m²) is used. The operating pressure is set at 0.25 MPa, and the temperature is controlled at 15-20℃. The supernatant is passed through the ultrafiltration membrane at a flow rate of approximately 160 mL / min. The permeate is collected to remove incompletely enzymatically hydrolyzed large protein molecules (molecular weight > 10 kDa). The retentate (approximately 700 mL, containing large protein molecules) is discarded, and approximately 4100 mL of permeate enters the second-stage ultrafiltration.

[0151] Second-stage ultrafiltration: A polyethersulfone ultrafiltration membrane with a molecular weight cutoff of 3 kDa (effective membrane area 1.0 m²) is used. The operating pressure is set at 0.20 MPa, and the temperature is controlled at 15-20℃. The first-stage permeate is passed through the ultrafiltration membrane at a flow rate of approximately 130 mL / min. The permeate (approximately 3100 mL, mainly consisting of small molecule amino acids and peptides with a molecular weight <3 kDa) is discarded, and the retentate is collected.

[0152] Continue ultrafiltration to concentrate the retentate. Stop ultrafiltration when the volume is concentrated to about 1000 mL (the concentration factor is about 5 times, relative to the volume of permeate before the second stage of ultrafiltration). 1000 mL of collagen peptide concentrate with a molecular weight of 3-10 kDa is obtained. It is a brownish-yellow viscous liquid with a solid content of about 17%.

[0153] e) Removing bitterness and fishy smell, and drying:

[0154] Transfer 1000 mL of collagen peptide concentrate to a reaction vessel and cool to 40°C. Add 3.15 g of debittering exopeptidase (enzyme activity 100 U / mg) (0.3% of the concentrate mass, which is approximately 1050 g based on a density of 1.05 g / mL). Gently stir (100 rpm) for 30 min. After debittering, rapidly heat the reaction solution to 95°C and hold for 10 min to inactivate the enzyme, then cool to room temperature.

[0155] Add 21.0 g of activated carbon (2.0% of the concentrate mass) to the cooled concentrate and stir for adsorption for 30 min (150 rpm). During the adsorption process, simultaneously introduce high-purity nitrogen gas (99.99% purity) at a flow rate of 300 mL / min, uniformly dispersed in the liquid through an aeration head, and perform nitrogen replacement for 30 min to remove dissolved oxygen and volatile fishy-smelling substances.

[0156] After adsorption and nitrogen replacement, the activated carbon is removed by filtration with multiple layers of gauze and filter paper, resulting in a concentrated solution of about 950 mL after debittering and deodorizing treatment. The solution is a light yellow, clear liquid with a characteristic slight fishy aroma and no obvious bitterness or fishy smell.

[0157] Drying process: Considering the efficiency requirements of industrial production, spray drying is adopted.

[0158] 950 mL of the concentrated solution after debittering and deodorizing treatment was sent to a spray dryer (LPG-5 type) for spray drying. The operating parameters were set as follows:

[0159] Feed rate: 40L / h (the concentrate is diluted to a suitable concentration).

[0160] Inlet air temperature: 140℃

[0161] Air outlet temperature: 80℃

[0162] Atomizer speed: 22000 rpm

[0163] Atomization pressure: 2.5MPa

[0164] The spray drying process takes approximately 24 minutes, yielding 165g of eel bone collagen peptide powder. The powder is a pale yellow, fine powder with a particle size of 60-100μm and a bulk density of approximately 0.42 g / cm³. 3 It has excellent liquidity.

[0165] Comparative Example 1: Without acid deashing, the aggregate after ethanol defatting was directly enzymatically hydrolyzed, and the remaining steps and conditions were the same as in Example 1.

[0166] Comparative Example 2: The three-stage complex enzymatic hydrolysis was not used; only neutral protease was used for single enzymatic hydrolysis (keeping the total enzyme activity the same). The remaining steps and conditions were the same as in Example 1.

[0167] Comparative Example 3: In the second enzymatic hydrolysis process, ultrasonic pulse treatment was not used; instead, the traditional enzymatic hydrolysis method was employed. The remaining steps and conditions were the same as in Example 1.

[0168] The performance of the eel bone collagen peptide powders prepared in the examples and comparative examples was evaluated:

[0169] Physicochemical indicators and immunomodulatory activity were tested: protein content was determined by the Kjeldahl method (GB 5009.5), molecular weight distribution and purity were determined by gel permeation chromatography (SEC-HPLC), water solubility was determined by solubility assay, and immunomodulatory activity was evaluated by ConA-induced mouse spleen lymphocyte proliferation assay (MTT method) and RAW264.7 macrophage activation assay (NO release assay, Griess method).

[0170] Immunomodulatory activity was evaluated using a ConA-induced mouse spleen lymphocyte proliferation assay. Spleens were harvested from male BALB / c mice (6-8 weeks old) under aseptic conditions, and spleen lymphocyte suspensions were prepared, with the cell concentration adjusted to 5 × 10⁻⁶ cells / mL. 6 Cells / mL. Cell suspension was seeded into 96-well plates (100 μL per well), and different concentrations of sample solution (final concentrations of 0, 50, 100, and 200 μg / mL) and ConA (final concentration of 5 μg / mL) were added. The plates were incubated at 37°C and 5% CO2 for 48 hours. Four hours before the end of the incubation period, MTT solution (5 mg / mL, 20 μL per well) was added, and the plates were incubated for another 4 hours. The supernatant was discarded, and 150 μL of DMSO was added to each well. The plates were shaken for 10 minutes to fully dissolve the crystals, and the absorbance was measured at 570 nm using a microplate reader. The proliferation rate of each group was calculated with the control group (without sample) as 100%. Each concentration was tested in 6 replicates, and the experiment was repeated 3 times.

[0171] The activation effect of the samples on macrophages was evaluated using the RAW264.7 mouse macrophage cell line. Cells were seeded in 96-well plates (5 × 10⁶ cells per well). 4Add sample solutions of different concentrations (final concentrations of 0, 50, 100, and 200 μg / mL) to each cell / 100 μL, and incubate at 37°C and 5% CO2 for 24 hours. Take 50 μL of the supernatant and add an equal volume of Griess reagent (a mixture of 1% sulfonamide and 0.1% N-1-naphthylethylenediamine dihydrochloride), react at room temperature for 10 minutes, and measure the absorbance at 540 nm using a microplate reader. Establish a standard curve using sodium nitrite standard solution and calculate the NO release (μM). Each concentration has 6 replicates, and the experiment is repeated 3 times.

[0172] (2) Stability Test: The eel bone collagen peptide powders prepared in each example and comparative example were stored for 6 months at 40℃±2℃ and relative humidity of 75%±5%, packaged in triple-layer composite aluminum foil bags (oxygen permeability ≤0.3cc / m). 2 •day), regularly test protein content, molecular weight distribution, color changes, turbidity and microbial indicators.

[0173] Table 1 Comparison of Physicochemical Indicators and Immunological Activity

[0174]

[0175] Lymphocyte proliferation rate: Sample concentration 100 μg / mL, ConA concentration 5 μg / mL, cultured for 48 h, with the blank control group as 100%. NO release: Sample concentration 200 μg / mL, stimulated RAW264.7 cells for 24 h, NO release in the blank control group was 8.2±0.8 μM.

[0176] Table 2. Accelerated stability test results (40℃, 75%RH, 6 months)

[0177]

[0178] ΔE: Color difference value, ΔE<5 indicates slight change, 5<ΔE<10 indicates significant change. Immunological activity retention rate: calculated based on lymphocyte proliferation rate.

[0179] In Comparative Example 1, the lack of acid deashing treatment resulted in a significant decrease in enzymatic hydrolysis efficiency. The main components of eel bone include collagen and hydroxyapatite, which are tightly bound together to form a complex matrix structure. Without deashing, numerous mineral crystals surround the collagen fibers, forming a dense physical barrier that severely hinders effective contact between protease molecules and the collagen substrate. Simultaneously, high concentrations of calcium and phosphate ions interfere with the conformational stability of the enzyme protein, reducing enzyme activity and affecting the pH buffering capacity of the reaction system, causing the pH to deviate from the optimal range for protease activity, thus significantly reducing enzymatic hydrolysis efficiency.

[0180] Comparative Example 2 showed that single-enzyme hydrolysis was significantly less effective than three-stage combined enzymatic hydrolysis. Collagen possesses a unique triple helix structure and a complex cross-linking network. A single protease has limited cleavage sites and cannot fully open up the hierarchical structure of collagen. In contrast, three-stage combined enzymatic hydrolysis achieves complementary advantages through the synergistic action of different enzymes: neutral protease performs initial coarse cleavage, disrupting the primary structure of collagen; Asp-N protease specifically cleaves the N-terminus of aspartic acid residues, directionally generating active peptides rich in acidic amino acids; and flavor protease further modifies the peptide chain ends, optimizing sensory properties. This sequential enzymatic hydrolysis strategy can more comprehensively hydrolyze collagen, producing active peptides with a more rational molecular weight distribution.

[0181] Comparative Example 3 showed that the enzymatic hydrolysis efficiency without ultrasonic pulse-assisted treatment was lower than that with ultrasonic-assisted treatment. The mechanical vibration and cavitation effect of ultrasound can disrupt the hydrogen bond network and hydrophobic interactions of collagen fiber bundles, increasing the swelling degree and porosity of the matrix, thereby improving the permeability of enzyme molecules and the accessibility of the substrate. Furthermore, the microjets and localized high-temperature and high-pressure environment generated by ultrasonic treatment can promote the formation of enzyme-substrate complexes, accelerate the reaction kinetics, and achieve higher hydrolysis degree and better peptide distribution within the same time frame.

[0182] The above description is only a preferred embodiment of the present invention, but the scope of protection of the present invention is not limited thereto. Any equivalent substitutions or modifications made by those skilled in the art within the scope of the technology disclosed in the present invention, based on the technical solution and inventive concept of the present invention, should be covered within the scope of protection of the present invention.

Claims

1. A method of preparing an immunity-enhancing eel bone peptide composition, characterized by, Includes the following steps: a) Pretreatment: The eel bone material is successively subjected to low-temperature freeze drying, crushing to below 200 mesh, ethanol degreasing and acid deashing to obtain pretreated bone material; b) Three-stage compound enzymatic hydrolysis: The pretreated aggregate is subjected to three-stage compound enzymatic hydrolysis to obtain the enzymatic hydrolysate; c) Enzyme inactivation and centrifugation: After inactivating the enzyme in the enzyme hydrolysate, centrifuge to remove insoluble matter and obtain a clear liquid; d) Ultrafiltration separation: The clear liquid is subjected to ultrafiltration separation to remove macromolecular impurities and obtain collagen peptide concentrate; e) Debittering and deodorizing and drying: The collagen peptide concentrate is debittered and deodorized, and then dried to obtain eel bone collagen peptide powder. The conditions for acid deashing are as follows: the degreased aggregate is treated with a 0.3-0.5 mol / L lactic acid solution at a mass ratio of 1:(8-10) and stirred at 4-8℃ for 2-4 hours; during the deashing process, samples are taken every 30 minutes to detect the ash content, and deashing is stopped when the ash content drops to 6.5-8.0%; The specific parameters for the three-stage complex enzymatic hydrolysis described in step b) are as follows: First stage: Add neutral protease, adjust pH to 7.0, temperature 50-55℃, and hydrolyze for 2 hours; Second stage: Add Asp-N protease, adjust pH to 7.5-8.5, temperature 40-45℃, and hydrolyze for 4-6 hours; Third stage: Add flavor protease, adjust pH to 6.5-7.0, temperature 45-50℃, and hydrolyze for 1-2 hours; The material-to-liquid mass ratio is maintained at 1:(8-12) during each stage of enzymatic hydrolysis. Step b) The second enzymatic hydrolysis process is combined with ultrasonic pulse treatment. The ultrasonic treatment is carried out throughout the entire second enzymatic hydrolysis process: the first half uses a frequency of 40kHz, 350W, working for 3 seconds and pausing for 1 second; the second half uses a frequency of 20kHz, 280W, working for 2 seconds and pausing for 4 seconds. The enzyme inactivation conditions described in step c) are a temperature of 95°C and a time of 10 min; the centrifugation conditions are a temperature of 4°C, a rotation speed of 4000-6000 rpm, and a time of 15-20 min. In step d), the ultrafiltration separation is carried out in two stages: the first stage uses an ultrafiltration membrane with a molecular weight cutoff of 10 kDa and an operating pressure of 0.15-0.25 MPa to collect the permeate and remove large molecular weight proteins. The second stage uses an ultrafiltration membrane with a molecular weight cutoff of 3 kDa, operating at a pressure of 0.10-0.20 MPa. The retentate is collected and concentrated 3-5 times to obtain a collagen peptide concentrate with a molecular weight of 3-10 kDa. In step e), the collagen peptide concentrate is cooled to 35-40℃, and debittering exopeptidase with an enzyme activity of 50-100 U / mg is added at a rate of 0.1-0.3% of the concentrate mass. The mixture is treated for 30 min, and then activated carbon at a rate of 1-2% of the concentrate mass is added for adsorption. At the same time, nitrogen gas is passed through at a flow rate of 200-300 mL / min for purging for 30 min. After filtering to remove the activated carbon, the mixture is dried using spray drying or freeze drying.

2. The method of claim 1, wherein the method of preparing the enhanced immunity eel bone peptide composition is characterized by, The conditions for ethanol degreasing in step a) are: degreasing temperature 4-8℃, degreasing time 2-4h, and material-to-liquid mass ratio 1:(3-5).

3. The method for preparing an immune-enhancing eel bone peptide composition according to claim 1, characterized in that, In the compound enzyme, the mass ratio of neutral protease, Asp-N protease and flavor protease is (1-1.7):(2.7-4):(0.7-1.3), and the total amount of compound enzyme added is 1.3-1.9% of the mass of pretreated aggregate.

4. The method for preparing an immune-enhancing eel bone peptide composition according to claim 1, characterized in that, The eel bone material is a by-product of processing fresh eel, with a water content controlled below 10% and a protein content ≥15%.

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