A two-stage controlled enzymatic preparation method of a ricefield eel bioactive peptide, and a product and application thereof

Abstract

The application discloses a two-stage controlled enzymolysis preparation method of a rice field eel bioactive peptide, and a product and application thereof, and relates to the technical field of food biotechnology and deep processing of aquatic resources, and comprises the following steps: homogenizing and adding water to the pretreated rice field eel, and performing hot treatment by adding ascorbic acid; after cooling the material, pH is adjusted to 8.0-8.5, alkaline protease is added for first-stage enzymolysis, and the system hydrolysis degree is monitored; when the system hydrolysis degree reaches 12%-15%, without intermediate enzyme inactivation, a compound protease is added for second-stage enzymolysis, the compound protease simultaneously contains aminopeptidase activity and carboxypeptidase activity; after the second-stage enzymolysis is completed, the enzyme is inactivated, and then insoluble solids are removed; the obtained supernatant is separated through an ultrafiltration membrane, the permeate is collected, desalination is performed, and drying is performed, so that a rice field eel bioactive peptide product is obtained; the rice field eel bioactive peptide product prepared by the application has the characteristics of low trimethylamine, low bitterness, stable molecular weight distribution and good quality consistency.

Description

Technical Field

[0001] This invention relates to the fields of food biotechnology and deep processing of aquatic resources, specifically to a two-stage controlled enzymatic hydrolysis method for preparing bioactive peptides from eels, and the resulting products and their applications. Background Technology

[0002] The swamp eel (Monopterus albus) is a freshwater economic fish with a high protein content and a relatively balanced amino acid composition, making it a potential raw material for the preparation of bioactive peptides. Unlike general terrestrial animal protein raw materials and poultry egg protein raw materials, the swamp eel raw material system has two unique problems: First, it contains a certain amount of trimethylamine oxide (TMAO), which is easily converted into trimethylamine (TMA) under the action of endogenous enzymes or microorganisms during processing, resulting in a noticeable fishy smell; second, the proportion of hydrophobic amino acids in swamp eel muscle is relatively high, and during deep enzymatic hydrolysis (especially when hydrophobic amino acids exist in free or exposed peptide terminus forms), bitter peptides are easily formed in the product.

[0003] In existing processes for preparing bioactive peptides from aquatic proteins, single-protease hydrolysis is commonly used, such as hydrolysis with alkaline protease as the main enzyme for a relatively long time. This results in a difficult-to-control molecular weight distribution of the product, with a high proportion of bitter peptides and free amino acids. Other processes employ stepwise hydrolysis using endopeptidases and exopeptidases. The common practice is to inactivate the enzyme by raising the temperature after the first stage of hydrolysis, followed by adjusting the pH and temperature for the second stage. While this process can modify peptide ends to some extent, the intermediate high-temperature enzyme inactivation process easily exacerbates the formation of trimethylamine-containing flavor defects, and the enzyme from the first stage is completely inactivated in the second stage, failing to exert a synergistic effect.

[0004] It is evident that existing aquatic protein bioactive peptides suffer from problems such as high trimethylamine content, numerous bitter peptides, and wide molecular weight distribution. Furthermore, existing preparation processes cannot simultaneously meet the requirements of inhibiting the conversion of eel characteristic fishy odor precursors, maintaining stable molecular weight distribution, and suppressing bitterness. Summary of the Invention

[0005] This invention aims to address the technical problem that existing aquatic protein bioactive peptide preparation processes cannot simultaneously achieve the inhibition of precursor conversion of characteristic fishy odor in eels, stable molecular weight distribution, and inhibition of bitterness. The goal is to provide a two-stage controlled enzymatic hydrolysis preparation method for eel bioactive peptides, along with its products and applications. This method combines ascorbic acid-assisted heat treatment, hydrolysis degree monitoring with rapid pH switching, continuous enzymatic hydrolysis using indestructible enzymes to link the two stages of enzymatic hydrolysis, and the selection of a complex protease containing both aminopeptidase and carboxypeptidase activity for the second stage of enzymatic hydrolysis. This results in eel bioactive peptide products that simultaneously possess low trimethylamine, low bitterness, stable molecular weight distribution, and good quality consistency.

[0006] This invention is achieved through the following technical solution:

[0007] The first objective of this invention is to provide a two-stage controlled enzymatic hydrolysis method for preparing bioactive peptides from swamp eels, comprising the following steps:

[0008] (1) After pretreatment, the eel is homogenized and water is added. 0.08% to 0.12% ascorbic acid is added according to the weight of the eel raw material. The mixture is then heat-treated at 90℃ to 95℃ for 12 to 18 min.

[0009] (2) After cooling the material obtained in step (1), adjust the pH to 8.0 to 8.5, add 0.8% to 1.2% alkaline protease at 50℃ to 55℃ according to the weight of the raw eel for the first stage of enzymatic hydrolysis, and monitor the degree of hydrolysis of the system;

[0010] (3) When the degree of hydrolysis of the system in step (2) reaches 12% to 15%, without intermediate enzyme inactivation, the pH of the system is adjusted to 6.0 to 6.5 within 10 min, and 0.3% to 0.7% of the complex protease is added according to the weight of the raw eel. The second stage of enzymatic hydrolysis is carried out at 45℃ to 55℃ for 3 to 4 h. The complex protease contains both aminopeptidase activity and carboxypeptidase activity.

[0011] (4) After the second stage of enzymatic hydrolysis is completed, the enzyme is inactivated, and then the insoluble solids are removed (by centrifugation or filtration);

[0012] (5) Separate the supernatant obtained in step (4) by ultrafiltration membrane (the molecular weight cutoff of the ultrafiltration membrane is preferably 3000-5000 Da), collect the permeate, desalt and dry it to obtain the eel bioactive peptide product.

[0013] The technical principle of this invention is as follows:

[0014] First, ascorbic acid is added during the homogenization stage, and synergistic heat treatment is carried out at a specific temperature of 90℃~95℃. The heat treatment inactivates the endogenous enzymes and trimethylamine-producing microorganisms in the eel raw material, reducing the amount of trimethylamine produced from the source. During the heat treatment, ascorbic acid consumes dissolved oxygen in the system, inhibits the proliferation of aerobic trimethylamine-producing microorganisms, and reduces the oxidation potential of the system, inhibiting the oxidative degradation of unsaturated lipids during subsequent enzymatic hydrolysis. Therefore, the synergistic effect of ascorbic acid and heat treatment at 90℃~95℃ reduces the amount of trimethylamine produced and reduces the fishy smell.

[0015] Then, the enzymatic hydrolysis process is carried out. During the first stage of enzymatic hydrolysis, the degree of hydrolysis (DH) value of the system is monitored, which can accurately identify the preset DH threshold and promptly trigger the pH switching operation to enter the second stage of enzymatic hydrolysis. This ensures the timeliness of threshold judgment and avoids batch-to-batch differences caused by simply controlling the pH based on time. The pH switching process is strictly controlled within 10 minutes (preferably within 5 minutes) to avoid hydrolysis overshoot caused by the enzyme acting for a long time in an unsuitable pH range in the first stage, which would lead to fluctuations in the molecular weight distribution of the product. Therefore, by using hydrolysis degree monitoring in combination with rapid pH switching, compared with simply controlling the pH based on time or long-term pH adjustment, the risk of hydrolysis overshoot can be significantly reduced, and the stability of molecular weight distribution and product quality consistency can be improved.

[0016] Furthermore, the first-stage enzymatic hydrolysis utilizes alkaline protease to break down large protein molecules into medium-molecular-weight peptides, providing a suitable substrate for the second-stage enzymatic hydrolysis. After the first-stage enzymatic hydrolysis is completed, a continuous enzymatic hydrolysis method without enzyme inactivation is used to connect the two stages of enzymatic hydrolysis. This allows the alkaline protease from the first stage to retain residual activity under the conditions of the second stage and to work together with the complex protease. This avoids the increased energy consumption and secondary increase in trimethylamine caused by intermediate high-temperature enzyme inactivation. Therefore, compared with the conventional intermediate enzyme inactivation stepwise enzymatic hydrolysis method, the present invention significantly reduces the trimethylamine content of the product and reduces process energy consumption.

[0017] Furthermore, the enzyme used in the second stage of enzymatic hydrolysis in this invention is a complex protease, which contains both aminopeptidase and carboxypeptidase activities. The complex protease gradually removes hydrophobic amino acid residues from the ends of the already formed medium molecular weight peptides. Compared with the enzymatic hydrolysis method using only a single carboxypeptidase, it can more systematically modify the ends of peptides, reduce the proportion of bitter peptides, and make the molecular weight distribution of the product more concentrated in the range of 500 to 1500 Da.

[0018] The aforementioned end-modification effects were verified through blind sensory evaluation: the bitterness intensity score of Example 1 (complex protease) was 1.2±0.3, while the bitterness intensity score of Comparative Example 5 (without exopeptidase) was 2.3±0.4, with a statistically significant difference (P < 0.05), directly demonstrating the technical advantage of the synergistic effect of the two exopeptidases in reducing the perceived bitterness. Furthermore, the overall acceptability scores of Examples 1–3 were all ≥8.0, while the overall acceptability score of Comparative Example 2 (single-enzyme deep hydrolysis) was only 5.2±0.5, indicating the significant improvement in flavor quality achieved by the two-stage controlled enzymatic hydrolysis strategy of this invention.

[0019] In summary, the method of this invention synergistically reduces trimethylamine formation and fishy odor through ascorbic acid and heat treatment at 90℃~95℃. By combining hydrolysis degree monitoring with rapid pH switching, the risk of hydrolysis overshoot is significantly reduced, improving molecular weight distribution stability and product quality consistency. The two-stage enzymatic hydrolysis is connected by continuous enzymatic hydrolysis with non-inactivating enzymes, allowing alkaline protease and complex protease to work together, avoiding secondary increases in trimethylamine caused by intermediate high-temperature enzyme inactivation, and significantly reducing trimethylamine content. By selecting a complex protease containing both aminopeptidase and carboxypeptidase activity for the second-stage enzymatic hydrolysis, the molecular weight distribution of the product is more concentrated in the 500~1500 Da range, achieving the preparation of eel bioactive peptides with low trimethylamine, low bitterness, and stable molecular weight distribution.

[0020] Furthermore, in step (1), the heat treatment is 92℃~94℃ and the heat treatment time is 14~16 min.

[0021] Further, in step (2), the alkaline protease is selected from one or more of the following: alkaline protease from Bacillus subtilis, alkaline protease from Bacillus licheniformis, and alkaline protease from Bacillus alkaliphilus.

[0022] Furthermore, in step (2), the degree of hydrolysis (DH) is monitored using the pH-stat method, the OPA colorimetric method, or the formaldehyde titration method.

[0023] Hydrolysis (DH) monitoring is preferably performed online using the pH-stat method. The pH-stat method uses an automated titration system to record the volume of alkali consumed in real time during enzymatic hydrolysis to maintain a constant pH. Based on the stoichiometric relationship between alkali consumption and peptide bond breakage, the degree of hydrolysis (DH) is continuously calculated. This method provides real-time DH values ​​without the need for sampling, enabling accurate identification of the preset DH threshold and timely triggering of the pH switching operation to enter the second stage of enzymatic hydrolysis. Alternatively, the OPA colorimetric method or formaldehyde titration method can be used for DH monitoring. When using offline detection methods, the detection interval is preferably no more than 15 minutes, and more preferably no more than 10 minutes, to ensure timely DH threshold determination. When the most recent offline detection result shows that DH has reached or exceeded the preset threshold, the pH switching operation is immediately initiated.

[0024] Further, in step (3), the complex protease is selected from one or more of the following: a complex protease preparation derived from Aspergillus oryzae, a complex protease preparation derived from Aspergillus niger, and a complex protease preparation derived from Bacillus subtilis; the complex protease contains both aminopeptidase activity and carboxypeptidase activity (preferably a complex protease preparation derived from Aspergillus oryzae, which contains both aminopeptidase and carboxypeptidase active components, such as Nanning Dongheng Huadao flavor enzyme or similar commercial food-grade complex protease products).

[0025] Further, in step (3), the pH of the system is adjusted to 6.0 to 6.5 within 10 min, preferably within 5 min, using phosphate buffer or citrate-disodium hydrogen phosphate buffer with a concentration of 30 to 150 mmol / L.

[0026] Further, in step (4), the enzyme hydrolysate is heated to 95℃~100℃ and held for 8~12 min to inactivate the enzyme.

[0027] Further, in step (5), desalination is performed using nanofiltration membrane desalination or ion exchange resin desalination, and drying is performed using vacuum freeze drying or low-temperature spray drying. The vacuum freeze drying conditions are preferably: pre-freezing temperature -45℃ to -25℃, more preferably -40℃ to -30℃; pre-freezing time 3 to 8 h, more preferably 4 to 6 h; vacuum degree during sublimation drying stage 5 to 80 Pa, more preferably 10 to 50 Pa; and temperature during desorption drying stage 15℃ to 30℃, more preferably 20℃ to 25℃.

[0028] The second objective of this invention is to provide a swamp eel bioactive peptide product, which is prepared by the method described in any one of claims 1 to 8, and the swamp eel bioactive peptide product meets the following indicators:

[0029] (1) Peptides with a molecular weight below 3000 Da account for more than 85%;

[0030] (2) Peptides with a molecular weight in the range of 500 to 1500 Da account for 35% to 65% of the total.

[0031] (3) The trimethylamine content is less than 1.2 mg / kg;

[0032] (4) Total protein and polypeptide dry basis content ≥90%;

[0033] (5) The total content of alanine, valine, methionine, isoleucine, leucine, proline, and phenylalanine in the product accounts for 30% to 40% of the total amino acid mass. The content of free amino acids is less than 10% of the total nitrogen, preferably less than 8%; the proportion of hydrophobic amino acids (Ala, Val, Leu, Ile, Pro, Phe, Trp, Met) in the total amino acids is 30% to 50%, preferably 30% to 40%.

[0034] A third objective of this invention is to provide the application of the aforementioned eel bioactive peptide product in the preparation of solid beverages, protein peptide nutritional products, or food ingredients. Preferably, the amount of the product added to solid beverages or protein peptide nutritional products is 3% to 30% (by weight), more preferably 5% to 20%.

[0035] Compared with the prior art, the present invention has the following advantages and beneficial effects:

[0036] 1. The method of this invention synergistically reduces the formation of trimethylamine and decreases fishy odor through ascorbic acid and heat treatment at 90℃~95℃. By combining hydrolysis degree monitoring with rapid pH switching, the risk of hydrolysis overshoot is significantly reduced, improving the stability of molecular weight distribution and product quality consistency. The two-stage enzymatic hydrolysis is connected by continuous enzymatic hydrolysis with non-inactivating enzymes, allowing alkaline protease and complex protease to work together, avoiding the secondary increase of trimethylamine caused by intermediate high-temperature enzyme inactivation, and significantly reducing trimethylamine content. By selecting a complex protease containing both aminopeptidase and carboxypeptidase activities for the second-stage enzymatic hydrolysis, the molecular weight distribution of the product is more concentrated in the 500~1500 Da range. The prepared eel bioactive peptide product has the characteristics of low trimethylamine, low bitterness (blind sensory evaluation panel bitterness intensity ≤1.5 points, overall acceptability ≥8.0 points), stable molecular weight distribution, and good quality consistency. It can be applied in the fields of solid beverages, protein peptide nutritional products, biopharmaceutical manufacturing, and food ingredients.

[0037] 2. This invention avoids excessive degradation caused by prolonged hydrolysis with a single peptidase by strictly controlling the degree of hydrolysis within a preferred window of 12%–15%. Furthermore, it employs a complex protease containing both aminopeptidase and carboxylpeptidase activities to synergistically cleave hydrophobic amino acid residues from both ends of the peptide, thereby inhibiting the formation of hydrophobic bitter peptides. It is well known in the art that the bitterness of protein hydrolysates mainly originates from the exposure of hydrophobic peptides due to excessive hydrolysis; however, the product obtained by this invention has a concentrated molecular weight distribution (see...). Figure 2 The proportion of short peptides can be controlled, which is beneficial for reducing bitterness and improving flavor in process design. Attached Figure Description

[0038] To more clearly illustrate the technical solutions of the exemplary embodiments of the present invention, the accompanying drawings used in the embodiments will be briefly described below. It should be understood that the following drawings only show some embodiments of the present invention and should not be considered as a limitation of the scope. For those skilled in the art, other related drawings can be obtained based on these drawings without creative effort. In the drawings:

[0039] Figure 1 This is the molecular weight distribution spectrum of the product in Example 1 of the present invention;

[0040] Figure 2 This is a comparison chart showing the percentage of molecular weight distribution below 3000 Da between Examples 1-3 and Comparative Examples 2-6 of the present invention. Detailed Implementation

[0041] To make the objectives, technical solutions, and advantages of the present invention clearer, the technical solutions in the embodiments of the present invention will be clearly and completely described below in conjunction with the embodiments. Obviously, the illustrative embodiments and descriptions of the present invention are only used to explain the present invention and are not intended to limit the present invention.

[0042] The embodiments of the present invention will be described in detail below. However, unnecessary details may be omitted. For example, detailed descriptions of well-known matters and repeated descriptions may be omitted. This is to avoid making the following description unnecessarily lengthy and to facilitate understanding by those skilled in the art.

[0043] The "scope" disclosed in this invention is defined in the form of a lower limit and an upper limit. A given scope is defined by selecting a lower limit and an upper limit, which define the boundaries of the specific scope. The scope defined in this way can include or exclude end values, and can be arbitrarily combined; that is, any lower limit can be combined with any upper limit to form a scope.

[0044] Unless otherwise specified, all embodiments and optional embodiments of the present invention can be combined with each other to form new technical solutions.

[0045] Unless otherwise specified, all technical features and optional technical features of this invention can be combined to form new technical solutions.

[0046] Unless otherwise specified, the terms "comprising" and "including" as used in this invention can be open-ended or closed-ended. For example, "comprising" and "including" can mean that other substances not listed may also be included, or that only the listed substances may be included.

[0047] Unless otherwise specified, all steps of the present invention may be performed sequentially or randomly, preferably sequentially. For example, the method includes steps (a) and (b), indicating that the method may include steps (a) and (b) performed sequentially, or it may include steps (b) and (a) performed sequentially. For example, the mention that the method may also include step (c) indicates that step (c) may be added to the method in any order. For example, the method may include steps (a), (b), and (c), or it may include steps (a), (c), and (b), or it may include steps (c), (a), and (b), etc.

[0048] The technical solution of the present invention will be further described in detail below with reference to the embodiments.

[0049] It should be noted that the flavor quality of the eel bioactive peptide products obtained in the embodiments and comparative examples of this invention was evaluated using a blind sensory evaluation panel. The evaluation results were closely related to the peptide molecular weight distribution and the degree of exposure of terminal hydrophobic amino acids. The evaluation method was carried out in accordance with GB / T16291.1-2012 "General Guidelines for the Selection, Training and Management of Sensory Analysis Evaluators" and GB / T12312-2012 "Determination of Taste Sensitivity in Sensory Analysis", and the specific steps are as follows:

[0050] (1) Selection and composition of evaluators

[0051] Eight evaluators (four men and four women, aged 22-35) who have passed the basic taste sensitivity test are needed. They must not smoke, have no history of chronic rhinitis or taste disorders, and have not consumed spicy or irritating foods or taken any medications affecting taste within two hours prior to the evaluation. All evaluators will receive pre-training and be familiar with the rules for using the 10-point rating scale.

[0052] (2) Sample preparation

[0053] Each sample to be evaluated was dissolved in purified water at a concentration of 5% (w / v) and equilibrated at room temperature (25±1℃) for 15 min before evaluation. Each sample volume was 30 mL, placed in an odorless transparent glass, and coded with a random three-digit number (e.g., 372, 815). The order of presentation was randomized to eliminate the order effect.

[0054] (3) Evaluation environment and process

[0055] Evaluations were conducted in separate sensory evaluation booths using standard natural light (D65 standard light source or equivalent natural light). Evaluators rinsed their mouths with room temperature purified water after each tasting and waited 2 minutes before evaluating the next sample. One replicate sample was included in each round of evaluation to verify the consistency of evaluators' scores.

[0056] (4) Rating scale

[0057] The following three dimensions will be scored using a 10-point scale:

[0058] Bitterness intensity 0 = no bitterness, 10 = extremely bitter; the lower the score, the weaker the bitterness. fishy smell intensity 0 = no fishy smell, 10 = extremely fishy smell; the lower the score, the weaker the fishy smell. Overall acceptability 0 = completely unacceptable, 10 = very acceptable; the higher the score, the better the overall flavor.

[0059] (5) Data statistics

[0060] Scores for each dimension of each sample are expressed as mean ± standard deviation. One-way ANOVA was used to compare the significance of differences between groups, with a significance level set at P < 0.05; if the differences were significant, Duncan's multiple comparison method was used for pairwise comparisons. All statistical analyses were performed using SPSS 26.0 software.

[0061] (6) Evaluation results validity criteria

[0062] An intragroup correlation coefficient (ICC) of ≥0.75 for replicate samples is considered to indicate good consistency in the evaluator's scoring, and the evaluation results for that round are valid; if ICC < 0.75, the evaluator should be retrained or removed and the results recalculated.

[0063] Furthermore, unless otherwise specified, the experimental methods used in the embodiments are conventional methods. Unless otherwise specified, the materials, reagents, methods, and instruments used are all conventional materials, reagents, methods, and instruments in the art, and can be obtained commercially by those skilled in the art.

[0064] Example 1

[0065] A two-stage controlled enzymatic hydrolysis method for preparing bioactive peptides from swamp eels includes the following steps:

[0066] (1) Take 100 kg of fresh eel, clean it and remove the inedible parts such as the internal organs and head, then homogenize it. Add 200 kg of water at a material-to-liquid ratio of 1:2, add 100 g of ascorbic acid (0.1% of the raw material weight), stir evenly and heat treat at 92℃ for 15 min.

[0067] (2) Cool the slurry obtained in step (1) to 52°C, adjust the pH to 8.2 with 2 mol / L NaOH solution, and add 1.0 kg of alkaline protease (from Bacillus subtilis, enzyme activity 200,000 U / g) (accounting for 1.0% of the raw material weight) to carry out the first stage of enzymatic hydrolysis. Maintain the temperature at 52°C during the enzymatic hydrolysis process and use the pH-stat method for online monitoring.

[0068] (3) When the DH value is detected to reach 13.5%, the pH of the system is immediately adjusted to 6.2 within 8 min using 50 mmol / L phosphate buffer. 0.5 kg of complex protease (0.5% of the raw material weight) is added. The complex protease contains both aminopeptidase and carboxypeptidase active components (a complex protease preparation derived from Aspergillus oryzae, such as Nanning Dongheng Huadao Flavor Enzyme or similar commercial food-grade complex protease products) is selected. The temperature is maintained at 52℃, and the second stage of enzymatic hydrolysis is carried out for 3.5 h.

[0069] (4) After the second stage of enzymatic hydrolysis, heat the system to 98°C and keep it for 10 min to inactivate the enzyme. Cool it to room temperature and centrifuge to remove insoluble matter.

[0070] (5) The supernatant was fractionated by a 5000 Da molecular weight cutoff ultrafiltration membrane, the permeate was collected, and then desalted by a nanofiltration membrane until the conductivity was less than 1.5 mS / cm. Finally, the product was obtained by vacuum freeze drying (pre-freezing at -35℃ for 4 h; sublimation drying at 20 Pa vacuum; desorption drying at 22℃).

[0071] Test results: such as Figure 1 As shown, peptides with a molecular weight below 3000 Da accounted for 88.6%, peptides in the range of 500 to 1500 Da accounted for 52.3%, trimethylamine content was 1.05 mg / kg, and total protein and polypeptide dry basis content was 91.8%.

[0072] Blind sensory evaluation results: bitterness intensity 1.2±0.3 points, fishy smell intensity 1.5±0.4 points, overall acceptability 8.4±0.5 points.

[0073] Of the above indicators, trimethylamine content was determined using the picric acid colorimetric method, molecular weight distribution was determined using gel permeation chromatography (GPC), and total protein and peptide content were determined using the Kjeldahl method. Sensory evaluation methods were as described above. Subsequent examples and comparative examples all used the same detection methods.

[0074] The amino acid composition of the eel bioactive peptide product obtained in Example 1 was analyzed, and the results are shown in Table 1.

[0075] Table 1. Amino acid composition analysis results of the eel bioactive peptide product in Example 1

[0076]

[0077] The test results in Table 1 show that the total amino acid content of the product is 831.396 mg / g, with relatively high contents of glutamic acid, lysine, aspartic acid, glycine, and arginine, at 110.302 mg / g, 97.072 mg / g, 81.583 mg / g, 77.270 mg / g, and 74.642 mg / g, respectively. Furthermore, based on alanine, valine, methionine, isoleucine, leucine, proline, and phenylalanine, the total content of hydrophobic amino acids is 260.337 mg / g, accounting for 31.31% of the total amino acid content, which falls within the 30%–40% range defined in the claims.

[0078] It is noteworthy that the total proportion of hydrophobic amino acids in this embodiment was not significantly increased compared to Comparative Example 5 (single carboxypeptidase), but the sensory score of Example 1 (8.4 points) was significantly better than that of Comparative Example 5 (7.7 points). This result indicates that the technical advantage of the present invention does not stem from reducing the total hydrophobic amino acid content, but rather from the systematic modification of peptide ends by the complex protease—by simultaneously cleaving hydrophobic residues from both the N-terminus (aminopeptidase) and C-terminus (carboxypeptidase), the proportion of hydrophobic amino acids exposed at the ends is reduced, thereby reducing the perceived bitterness without altering the total amino acid composition. Furthermore, the above results are consistent with the present invention's focus on concentrated molecular weight distribution, flavor improvement, and application in nutritional products.

[0079] Example 2

[0080] The procedure is the same as in Example 1, except that: in step (1), the amount of ascorbic acid is 0.08%, the heat treatment temperature is 93℃ and the time is 14 min; in step (2), the alkaline protease is derived from Bacillus licheniformis; in step (3), the DH trigger threshold is 12%, the pH switching time is 5 min, and the second stage of enzymatic hydrolysis is 3 h.

[0081] Test results: Peptides below 3000 Da accounted for 89.2%, peptides in the 500-1500 Da range accounted for 53.1%, trimethylamine content was 0.95 mg / kg, and total protein and polypeptide dry basis content was 92.1%. Sensory evaluation panel blind test results: bitterness intensity 1.0±0.2 points, fishy smell intensity 1.4±0.3 points, overall acceptability 8.6±0.4 points.

[0082] Example 3

[0083] The procedure was performed according to Example 1, with the difference that the DH trigger threshold was 15%, the second-stage enzymatic hydrolysis lasted 4 hours, and the amount of complex protease used was 0.7%.

[0084] Test results: Peptides below 3000 Da accounted for 90.3%, peptides in the 500-1500 Da range accounted for 58.4%, trimethylamine content was 1.10 mg / kg, and total protein and polypeptide dry basis content was 91.5%. Sensory evaluation panel blind test results: bitterness intensity 1.3±0.3 points, fishy smell intensity 1.6±0.4 points, overall acceptability 8.2±0.5 points.

[0085] As can be seen from the summary of key indicators of the products obtained in Examples 1 to 3, within the preferred process window defined by the present invention, the obtained products all maintain a high proportion of peptides below 3000 Da and a low trimethylamine content. The bitterness intensity of the sensory evaluation is controlled below 1.5 points, and the overall acceptability is above 8.0 points. This indicates that the two-stage controlled enzymatic hydrolysis route described in the present invention has good process adaptability and result stability.

[0086] Comparative Example 1

[0087] No ascorbic acid added

[0088] Except for step (1), which does not include ascorbic acid, the steps in this comparative example are the same as in Example 1.

[0089] Test results: 88.0% of the peptides were below 3000 Da, and the trimethylamine content rose to 4.6 mg / kg. Sensory evaluation panel blind test results: bitterness intensity 3.2±0.5 points, fishy smell intensity 4.8±0.6 points, overall acceptability 6.3±0.5 points.

[0090] The results showed that when ascorbic acid was not added, the trimethylamine content increased significantly compared to Example 1, by more than four times, demonstrating that the synergistic effect of ascorbic acid and heat treatment can effectively reduce the amount of trimethylamine generated.

[0091] Comparative Example 2

[0092] Single-enzyme deep enzymatic hydrolysis

[0093] This comparative example only uses alkaline protease to continuously hydrolyze to DH 20% (approximately 5.5 h) at pH 8.2 and 52°C. Subsequent steps are the same as in Example 1.

[0094] Test results: Peptides below 3000 Da accounted for 79.0%, and trimethylamine content was 6.8 mg / kg. Sensory evaluation panel blind test results: bitterness intensity 5.8±0.6 points, fishy smell intensity 3.5±0.5 points, overall acceptability 5.2±0.5 points.

[0095] Results analysis: This comparative example only used alkaline protease for single-enzyme deep hydrolysis. Compared with Example 1, the trimethylamine content increased significantly from 1.05 mg / kg to 6.8 mg / kg. The main reasons include: (1) The optimal pH of alkaline protease is 8.0-8.5. Under this pH condition, the endogenous trimethylamine oxidase (TMAOase) or microbial enzyme system of the eel may still maintain some activity and reduce trimethylamine oxidase (TMAO) to trimethylamine (TMA). However, Example 1 effectively inhibited this conversion reaction by switching the pH in two stages (down to 6.0-6.5); (2) The single-enzyme deep hydrolysis time is as long as 5.5 h. The hydrolysis system is kept at a low temperature under alkaline conditions for a long time, which provides a longer window for the chemical degradation of TMAO and microbial metabolism; (3) Although the synergistic effect of ascorbic acid and heat treatment in Example 1 also exists in the single-enzyme system, the oxidation potential of the system gradually rises during the long-term hydrolysis process, and the consumption of ascorbic acid weakens its protective effect.

[0096] Regarding the reasons for the obvious bitterness: (1) The alkaline protease is an endopeptidase, and its continuous action leads to non-selective cleavage of peptide bonds, producing a large number of small peptides (<500 Da) and free amino acids with exposed hydrophobic amino acids at their ends. These small peptide fragments are the main bitter substances; (2) The second-stage modification of the complex protease (aminopeptidase + carboxypeptidase) in Example 1 is missing, and the bitterness cannot be reduced by systematically removing hydrophobic residues from both ends of the peptide fragments; (3) As Figure 2 As shown, the proportion of peptides below 3000 Da in Comparative Example 2 (79.0%) was significantly lower than that in Example 1 (88.6%), indicating that excessive hydrolysis resulted in the aggregation of a large number of bitter peptides with extremely small molecular weights in the product, rather than the functional peptides of 500–1500 Da within the target range of this invention.

[0097] Comparative Example 3

[0098] Two-stage enzymatic hydrolysis with intermediate enzyme inactivation

[0099] In this comparative example, when the DH reached 13.5% in the first stage of enzymatic hydrolysis, the temperature was raised to 98°C and held for 10 min to inactivate the enzyme. After cooling to 50°C, the pH was adjusted to 6.2, and then a complex protease was added to continue enzymatic hydrolysis for 3.5 h. The rest was the same as in Example 1.

[0100] Test results: 84.2% of the peptides were below 3000 Da, and the trimethylamine content was 1.8 mg / kg. Blind sensory evaluation results: bitterness intensity 2.5±0.4 points, fishy smell intensity 2.2±0.4 points, overall acceptability 7.5±0.5 points.

[0101] The results showed that the trimethylamine content increased significantly by using the intermediate enzyme inactivation stepwise method, from 1.05 to 1.8 mg / kg, an increase of 16.7%, and the proportion of products below 3000 Da also decreased significantly.

[0102] Comparative Example 4

[0103] pH switching time is too long

[0104] Except for extending the pH adjustment time to 30 min in the second stage and using the OPA colorimetric method (detection interval 20 min) for DH monitoring, the other steps are the same as in Example 1.

[0105] Test results: 83.1% of the peptides were below 3000 Da, and the trimethylamine content was 1.6 mg / kg. Blind sensory evaluation results: bitterness intensity 2.8±0.5 points, fishy smell intensity 2.0±0.4 points, overall acceptability 7.2±0.5 points.

[0106] The results showed that when the inherent delay of offline detection was combined with slow pH switching, the reaction time of the enzyme in the first stage within the transitional pH range (approximately 7.0–7.5) was significantly prolonged, leading to a degree of hydrolysis exceeding the preset threshold (overshooting), a wider molecular weight distribution of the product, and a decrease in the proportion of peptides below 3000 Da. This demonstrates the necessity of combining the pH-stat method for online DH monitoring with rapid pH switching (≤10 min), which is an important condition for ensuring product quality stability.

[0107] Comparative Example 5

[0108] The second stage does not add exopeptidase.

[0109] In this comparative example, no exopeptidase preparation was added in the second stage. The process continued for 3.5 h solely based on the residual alkaline protease from the first stage and its residual endopeptidase activity at pH 6.2 and 52°C. The rest of the process was the same as in Example 1.

[0110] The optimal pH for alkaline protease is 8.0–8.5. At pH 6.2, its endopeptidase activity has significantly decreased (with residual activity of about 10%–20%), but it can still act slowly and continuously, causing the peptide to be further cleaved non-selectively into free amino acids and small peptides with exposed hydrophobic residues at the ends. Therefore, the targeted modification effect of the compound exopeptidase on the peptide ends in Example 1 cannot be demonstrated.

[0111] Test results: Peptides below 3000 Da accounted for 85.1%, peptides in the 500-1500 Da range accounted for 41.2%, and the proportion of hydrophobic amino acids was about 6 percentage points higher than in Example 1. Sensory evaluation blind test results: bitterness intensity 2.3±0.4 points, fishy smell intensity 1.8±0.3 points, overall acceptability 7.7±0.4 points.

[0112] The results showed that limiting the activity type of the enzyme used in the second stage (containing both aminopeptidase and carboxylpeptidase activities) played a crucial role in the concentration of product molecular weight distribution and the inhibition of bitterness. The reason for this is:

[0113] (1) Dual-terminal synergistic modification mechanism: The complex protease acts simultaneously on the N-terminus (aminopeptidase removes amino acid residues one by one from the N-terminus) and C-terminus (carboxypeptidase removes amino acid residues one by one from the C-terminus) of the peptide, achieving systematic pruning of the hydrophobic residues at both ends of the peptide. In contrast, this comparative example does not add any exopeptidase, but relies solely on the residual endopeptidase to continue non-selectively cleaving the peptide bonds inside the peptide, resulting in the generation of a large number of small peptides (<500 Da) and free amino acids with exposed hydrophobic amino acids at the ends. These small molecule peptides are the main source of bitter substances.

[0114] (2) Molecular weight distribution regulation mechanism: The synergistic effect of the exopeptidases gradually prunes both ends of medium molecular weight peptides (500-1500 Da), preventing excessive degradation into free amino acids (the content of free amino acids in Example 1 is less than 10% of total nitrogen) and avoiding the residue of larger peptides (>1500 Da), thus making the molecular weight distribution more concentrated in the target range. In this comparative example, no exopeptidases were added, resulting in the proportion of peptides in the 500-1500 Da range being only 41.2% (52.3% in Example 1), and the proportion of free amino acids was significantly increased, confirming the negative impact of the lack of terminal modification enzymes on the regulation of molecular weight distribution.

[0115] (3) Sensory evaluation verification: The bitterness intensity of Comparative Example 5 was 2.3±0.4 points and the overall acceptability was 7.7±0.4 points, both of which were significantly worse than that of Example 1 (bitterness intensity 1.2±0.3 points, overall acceptability 8.4±0.5 points, P<0.05). From the sensory perspective, it was verified that the lack of exopeptidase terminal modification directly led to incomplete bitterness inhibition.

[0116] Comparative Example 6

[0117] DH threshold deviation

[0118] Except for the second stage trigger DH being set to 25%, the other steps in this comparative example are the same as in Example 1.

[0119] Test results: Peptides below 3000 Da accounted for 80.4%, free amino acids were high, and the batch-to-batch molecular weight distribution fluctuated significantly. Sensory evaluation panel blind test results: bitterness intensity 3.5±0.7 points, fishy smell intensity 2.4±0.5 points, overall acceptability 6.8±0.6 points.

[0120] Results Analysis: In this comparative example, the second-stage trigger DH was set to 25%, which is significantly higher than the 12%–15% range specified in this invention. Under this condition, the excessive action of the first-stage alkaline protease will lead to: (1) the substrate peptides are cleaved too finely, and a large number of peptides have molecular weights below 500 Da or even converted into free amino acids. The second-stage complex protease loses suitable medium molecular weight substrates and cannot exert the directional enrichment effect in the 500–1500 Da range; (2) the alkaline protease has been acting for too long under pH 8.2 conditions (estimated to be more than 6 h), and the system's exposure time in the alkaline environment increases, which exacerbates the conversion of TMAO to TMA. At the same time, the degree of lipid oxidation is deepened, resulting in the accumulation of fishy and off-flavor substances; (3) due to the excessive fragmentation of the substrate, the aminopeptidase and carboxypeptidase activities of the complex protease lose the targeting of "end pruning", and the peptide degradation is random, resulting in increased batch-to-batch molecular weight distribution fluctuations (RSD>10%).

[0121] Among them, the adverse effects of high levels of free amino acids include: (1) reduced functionality: free amino acids do not have the bioactive sites and spatial conformations of peptides, and lose the specific functions of active peptides; (2) flavor deterioration: some free amino acids (such as leucine, valine, and phenylalanine) have a bitter taste, and their increased content directly aggravates the bitterness of the product; (3) increased hygroscopicity: free amino acids have higher hygroscopicity than peptides, which makes the product easy to absorb moisture and clump during storage, affecting product stability; (4) decreased economic efficiency: with the same protein raw material input, the yield of the target active peptide (500-1500 Da) decreases, and the unit cost increases; (5) poor batch-to-batch consistency: DH 25% has exceeded the window of the preferred first-stage DH threshold (12%-15%) and the synergistic effect of the second stage. The second-stage enzymatic hydrolysis substrate is too fragmented, and the end modification of the complex protease loses its directionality, resulting in a significant increase in the fluctuation of molecular weight distribution between batches.

[0122] The above results demonstrate that precise control of the DH trigger threshold (12%–15%) is a key process parameter for achieving stable molecular weight distribution and consistent product quality in this invention. Exceeding this range will lead to a comprehensive deterioration of the product's functionality, flavor, and stability.

[0123] The sensory evaluation results of the eel bioactive peptide products of each embodiment and comparative example are shown in Table 2.

[0124] Table 2. Summary of sensory evaluation results of the bioactive peptide products from the eel in each example and comparative example.

[0125]

[0126] Note: The rating is based on a 10-point scale. Lower scores for bitterness and fishiness intensity are better, and higher scores for overall acceptability are better. All data are the mean ± standard deviation of blind reviews from 8 evaluators.

[0127] As shown in Table 2, within the process window defined by this invention, the bitterness intensity of Examples 1-3 was controlled at ≤1.5 points, the overall acceptability was ≥8.0 points, and the standard deviation between batches was small (≤0.5 points), indicating that the two-stage controlled enzymatic hydrolysis process of this invention has good flavor stability. Comparative Example 1, due to the absence of ascorbic acid, showed a significantly increased fishy odor intensity (4.8 points), demonstrating the key role of ascorbic acid in synergistic heat treatment for fishy odor suppression. Comparative Example 2, due to deep enzymatic hydrolysis with a single enzyme, had the highest bitterness intensity (5.8 points), proving that the lack of end-modification of the double exopeptidase directly leads to bitterness degradation of the product. Comparative Example 5, using only a single carboxypeptidase, had a significantly higher bitterness intensity (2.3 points) than Example 1 (1.2 points), proving that the synergistic effect of aminopeptidase and carboxypeptidase is a necessary condition for reducing bitterness. The above sensory evaluation results, along with the molecular weight distribution data, trimethylamine content data, and hydrophobic amino acid ratio data of each sample, mutually corroborate each other, forming a complete chain of evidence for the "low trimethylamine, low bitterness" technical effect of this invention.

[0128] Example 4

[0129] Solid beverage applications

[0130] Take 10 kg of the eel bioactive peptide powder obtained in Example 1, mix it evenly with 20 kg of maltodextrin, 10 kg of fructooligosaccharide, 100 g of vitamin C, 50 g of citric acid and 200 g of natural flavoring to make 100 kg of solid beverage product.

[0131] The resulting solid beverage exhibited good solubility; 10 g dissolved in 200 mL of warm water within 30 seconds, with no obvious precipitation and a pale yellow color. Blind sensory evaluation results showed: bitterness intensity 1.0±0.2 points, fishy odor intensity 1.2±0.3 points, and overall acceptability 8.6±0.4 points.

[0132] Finally, it should be noted that the above specific embodiments are only used to describe the purpose, technical solution, and beneficial effects of the present invention in detail. It should be understood that the above description is only a specific implementation of the present invention and is not intended to limit the scope of protection of the present invention. Although the present invention has been described in detail with reference to the foregoing specific embodiments, those skilled in the art should understand that modifications can still be made to the technical solutions described in the foregoing embodiments, or equivalent substitutions or improvements can be made to some or all of the technical features. Such modifications, equivalent substitutions, and improvements do not cause the essence of the corresponding technical solutions to deviate from the scope of the technical solutions of the embodiments of the present invention, and they should all be covered within the scope of the claims and specification of the present invention.

Claims

1. A two-stage controlled enzymatic hydrolysis method for preparing bioactive peptides from swamp eels, characterized in that, Includes the following steps: (1) After pretreatment, the eel is homogenized and water is added. 0.08% to 0.12% ascorbic acid is added according to the weight of the eel raw material. The mixture is then heat-treated at 90℃ to 95℃ for 12 to 18 min. (2) After cooling the material obtained in step (1), adjust the pH to 8.0 to 8.5, add 0.8% to 1.2% alkaline protease at 50℃ to 55℃ according to the weight of the raw eel for the first stage of enzymatic hydrolysis, and monitor the degree of hydrolysis of the system; (3) When the degree of hydrolysis of the system in step (2) reaches 12% to 15%, without intermediate enzyme inactivation, the pH of the system is adjusted to 6.0 to 6.5 within 10 min, and 0.3% to 0.7% of the complex protease is added according to the weight of the raw eel. The second stage of enzymatic hydrolysis is carried out at 45℃ to 55℃ for 3 to 4 h. The complex protease contains both aminopeptidase activity and carboxypeptidase activity. (4) After the second stage of enzymatic hydrolysis is completed, the enzyme is inactivated and then the insoluble solids are removed; (5) The supernatant obtained in step (4) is separated by ultrafiltration membrane, the permeate is collected, desalted and dried to obtain the eel bioactive peptide product.

2. The two-stage controlled enzymatic hydrolysis method for preparing bioactive peptides from swamp eels according to claim 1, characterized in that, In step (1), the heat treatment is 92℃~94℃ and the heat treatment time is 14~16 min.

3. The two-stage controlled enzymatic hydrolysis method for preparing bioactive peptides from swamp eels according to claim 1, characterized in that, In step (2), the alkaline protease is selected from one or more of the following: alkaline protease from Bacillus subtilis, alkaline protease from Bacillus licheniformis, and alkaline protease from Bacillus alkaliphilus.

4. The two-stage controlled enzymatic hydrolysis method for preparing bioactive peptides from swamp eels according to claim 1, characterized in that, In step (2), the degree of hydrolysis is monitored by pH-stat method, OPA colorimetric method or formaldehyde titration method.

5. The two-stage controlled enzymatic hydrolysis method for preparing bioactive peptides from swamp eels according to claim 1, characterized in that, In step (3), the complex protease is selected from one or more of the following: a complex protease preparation derived from Aspergillus oryzae, a complex protease preparation derived from Aspergillus niger, and a complex protease preparation derived from Bacillus subtilis.

6. The two-stage controlled enzymatic hydrolysis method for preparing bioactive peptides from swamp eels according to claim 1, characterized in that, In step (3), when adjusting the pH of the system to 6.0 to 6.5, a phosphate buffer or a citrate-disodium hydrogen phosphate buffer with a concentration of 30 to 150 mmol / L is used.

7. The two-stage controlled enzymatic hydrolysis method for preparing bioactive peptides from swamp eels according to claim 1, characterized in that, In step (4), the enzyme hydrolysate is heated to 95℃~100℃ and held for 8~12 min to inactivate the enzyme.

8. The two-stage controlled enzymatic hydrolysis method for preparing bioactive peptides from swamp eels according to claim 1, characterized in that, In step (5), desalination is performed using nanofiltration membrane desalination or ion exchange resin desalination, and drying is performed using vacuum freeze drying or low-temperature spray drying.

9. A bioactive peptide product from eel, characterized in that, The eel bioactive peptide product is prepared by the method according to any one of claims 1 to 8, and the eel bioactive peptide product meets the following indicators: (1) Peptides with a molecular weight below 3000 Da account for more than 85%; (2) Peptides with a molecular weight in the range of 500 to 1500 Da account for 35% to 65% of the total. (3) The trimethylamine content is less than 1.2 mg / kg; (4) Total protein and polypeptide dry basis content ≥90%; (5) The total content of alanine, valine, methionine, isoleucine, leucine, proline and phenylalanine in the product accounts for 30% to 40% of the total amino acid mass.

10. The application of the eel bioactive peptide product as described in claim 9 in the preparation of solid beverages, protein peptide nutritional products, or food ingredients.

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