Method for extracting active polypeptide from freshwater fish
Through the synergistic effect of the complex enzyme preparation and the bitter mandelic acid mixture, combined with the activation of the phenolic hydroxyl groups of aromatic amino acids by nitrogen infusion, the problems of insufficient polypeptide molecular weight control and antioxidant capacity in the extraction of active polypeptides from freshwater fish were solved, and an efficient and safe polypeptide extraction process was achieved.
Patent Information
- Application Number
- CN202511122547.3
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2025-08-12
- Publication Date
- 2025-09-26
- Estimated Expiration
- Not applicable · inactive patent
AI Technical Summary
Among the existing methods for extracting active peptides from freshwater fish, single enzymatic hydrolysis makes it difficult to accurately control the molecular weight of the peptide, resulting in a low proportion of the target product and insufficient antioxidant capacity. Mechanical crushing can easily cause thermal denaturation of the protein, and chemical infiltration methods can easily leave harmful substances and a high risk of heavy metal precipitation.
The synergistic effect of a complex enzyme preparation and a mixture of mandelic acid is used, combined with nitrogen gas infusion to activate the phenolic hydroxyl groups of aromatic amino acids, control the molecular weight distribution of polypeptides, and avoid protein denaturation through an enzymatic hydrolysis process under mild conditions, thereby improving polypeptide yield and antioxidant capacity.
It achieves efficient extraction of active peptides with high bioavailability, enhances antioxidant capacity, increases peptide yield and functional amino acid content, reduces raw material costs and environmental burden, and ensures the natural activity and safety of the product.
Smart Images
Figure CN120700091A_ABST
Abstract
Description
Technical Field
[0001] The invention belongs to the technical field of biological extraction, and particularly relates to a method for extracting active polypeptides from freshwater fish. Background Art
[0002] Currently, the extraction of active peptides from freshwater fish primarily relies on traditional enzymatic hydrolysis. A typical process involves using freshwater fish surimi or fish bones as raw material, homogenizing them, adding a single protease, hydrolyzing them at a constant temperature for a specific time, and then inactivating the enzyme and centrifuging them to obtain the peptide product. Existing technologies generally use mechanical crushing or chemical osmosis to pretreat the raw materials to improve extraction efficiency. Some methods attempt to add metal ion chelators to assist in calcium dissolution. The core goal of these processes is to degrade large molecular proteins into small molecular active peptides through enzymatic reactions.
[0003] However, in the above methods, single enzymatic hydrolysis makes it difficult to accurately control the molecular weight of the polypeptide, resulting in a low proportion of the target product, and lacks the directional activation of the phenolic hydroxyl groups of aromatic amino acids, resulting in insufficient antioxidant capacity. Mechanical crushing can easily cause thermal denaturation of the protein, while the chemical infiltration method is prone to residual harmful substances. Both reduce the polypeptide yield and increase the risk of heavy metal precipitation. Summary of the Invention
[0004] The object of the present invention is to provide a method for extracting active polypeptides from freshwater fish to solve the problems raised in the above background technology.
[0005] To achieve the above object, the present invention provides the following technical solutions:
[0006] A method for extracting active polypeptides from freshwater fish comprises the following steps:
[0007] (1) Take fresh freshwater fish bones, wash them and homogenize them;
[0008] (2) adding deionized water containing mandelic acid to the homogenate at a solid-liquid ratio of 3-5:1, wherein the deionized water contains 0.1-0.3 wt % of a mixture of D-mandelic acid and L-mandelic acid, and the weight ratio of D-mandelic acid to L-mandelic acid is 52-58:0.3-0.7;
[0009] (3) The mixture was stirred at a constant temperature of 47-52°C, the pH was adjusted to 6.7-7.0, and the complex enzyme preparation was added and enzymatically hydrolyzed for 4-6 hours;
[0010] (4) Nitrogen was introduced every 20-30 min during the enzymatic hydrolysis process at a rate of 15-25 g / h;
[0011] (5) After the enzymatic hydrolysis is completed, the enzymatic hydrolyzate is heated to 92-95°C and maintained for 8-12 minutes to inactivate the enzyme, the pH is adjusted to neutral, and then centrifuged at 4-10°C and 7000-8000 r / min for 10-15 minutes. The supernatant is collected to obtain the active polypeptide.
[0012] Preferably, the complex enzyme preparation consists of 4-10 parts of neutral protease, 3-5 parts of papain and 1-2 parts of trypsin.
[0013] Preferably, the active polypeptide has a molecular weight of 2000-3000 Da and contains aspartic acid, threonine, glutamic acid, glycine, alanine, isoleucine, tyrosine, phenylalanine, histidine, lysine and arginine.
[0014] Compared with the prior art, the present invention has the following beneficial effects:
[0015] (1) The present invention periodically introduces nitrogen during the enzymatic hydrolysis process to directionally activate the phenolic hydroxyl groups of aromatic amino acids in freshwater fish active polypeptides, making them efficient electron donors and enhancing the in vitro antioxidant capacity of the polypeptides; at the same time, the D / L-mandelic acid mixture synergistically acts on the cell membrane structure, improving cell permeability under mild conditions, avoiding protein denaturation caused by traditional mechanical crushing, and increasing the yield of active polypeptides and the content of functional amino acids.
[0016] (2) Through the synergistic catalysis of multiple enzymes in the complex enzyme system, the molecular weight distribution of the polypeptide is controlled to be in the ideal range, and the conversion and chelation of inorganic calcium from fish bones to organic calcium are simultaneously achieved to form a calcium chelating peptide with high bioavailability.
[0017] (3) The green enzymatic hydrolysis process achieves high-value utilization of waste resources, reducing raw material costs and environmental burdens; the process does not require high temperature, high pressure or strong chemical reagents, the reaction conditions are mild and there is no risk of reverse reaction, ensuring the natural activity and safety of the product. BRIEF DESCRIPTION OF THE DRAWINGS
[0018] Figure 1 It is a process flow chart of the present invention. DETAILED DESCRIPTION
[0019] The following will clearly and completely describe the technical solutions in the embodiments of the present invention in conjunction with the accompanying drawings. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without making creative efforts are within the scope of protection of the present invention.
[0020] Example 1:
[0021] See also Figure 1As shown, a method for extracting active polypeptides from freshwater fish comprises:
[0022] (1) Take 1 kg of fish bone scraps from the Yellow River carp, wash them and homogenize them;
[0023] (2) adding deionized water containing mandelic acid to the homogenate at a solid-liquid ratio of 3:1, wherein the deionized water contains 0.1 wt % of a mixture of D-mandelic acid and L-mandelic acid, and the weight ratio of D-mandelic acid to L-mandelic acid is 52:0.3;
[0024] (3) The mixture was stirred at a constant temperature of 47°C, the pH was adjusted to 6.7, and a composite enzyme preparation was added, which consisted of 4 parts of neutral protease + 3 parts of papain + 1 part of trypsin (0.8% by weight of fish bones), and enzymatic hydrolysis was carried out for 4 hours;
[0025] (4) During the enzymatic hydrolysis process, nitrogen was introduced every 30 min at a rate of 15 g / h;
[0026] (5) After the enzymatic hydrolysis is completed, the enzymatic hydrolyzate is heated to 92°C and maintained for 12 minutes to inactivate the enzyme, the pH is adjusted to neutral, and then centrifuged at 4°C and 7000 rpm for 15 minutes. The supernatant is collected to obtain the active polypeptide.
[0027] Example 2:
[0028] See also Figure 1 As shown, a method for extracting active polypeptides from freshwater fish comprises:
[0029] (1) Raw material processing: Take 1 kg of fish bone scraps from the Yellow River carp, wash them and homogenize them;
[0030] (2) adding deionized water containing mandelic acid to the homogenate at a solid-liquid ratio of 5:1, wherein the deionized water contains 0.3 wt % of a mixture of D-mandelic acid and L-mandelic acid, and the weight ratio of D-mandelic acid to L-mandelic acid is 58:0.7;
[0031] (3) The mixture was stirred at a constant temperature of 52°C, the pH was adjusted to 7.0, and a composite enzyme preparation was added, which consisted of 10 parts of neutral protease + 5 parts of papain + 2 parts of trypsin (added at 1.5% by weight of fish bones), and enzymatic hydrolysis was carried out for 6 hours;
[0032] (4) During the enzymatic hydrolysis process, nitrogen was introduced every 20 min at a rate of 25 g / h;
[0033] (5) After the enzymatic hydrolysis is completed, the hydrolyzate is heated to 95°C for 8 minutes to inactivate the enzyme, the pH is adjusted to neutral, and then centrifuged at 10°C and 8000 rpm for 10 minutes. The supernatant is collected to obtain the active polypeptide.
[0034] Example 2:
[0035] The performance comparison between the first embodiment and the second embodiment of the present invention and the prior art includes the following steps:
[0036] The experimental groups are as follows:
[0037]
[0038] The detection indicators and methods are as follows:
[0039]
[0040] The testing process includes:
[0041] (1) Sample preparation: Three groups were processed simultaneously. Except for the enzymatic hydrolysis conditions, other steps (homogenization, centrifugation, etc.) were completely consistent.
[0042] (2) Index detection:
[0043] Peptide yield: freeze-dry the supernatant and weigh it, and calculate: yield (%) = (peptide mass / fish bone mass) × 100;
[0044] Molecular weight distribution: GPC column (TSKgel G2000SWxl), mobile phase 0.1 M PBS, flow rate 0.5 mL / min;
[0045] DPPH scavenging rate: scavenging rate (%) = [1-(A_sample / A_control)] × 100 (A = absorbance at 517 nm);
[0046] Calcium chelation rate: chelation rate (%) = (organic calcium / total calcium) × 100 (dialysis molecular weight cut-off: 1000 Da); the test data are compared in the following table (data are the mean ± standard deviation of 3 repeated experiments):
[0047]
[0048] Nitrogen treatment increases the content of aromatic amino acids and the DPPH clearance rate, solving the problem of insufficient antioxidant properties in existing technologies.
[0049] A mixture of mandelic acids (specific D:L ratio) synergistically improves yield and avoids protein denaturation caused by traditional mechanical crushing.
[0050] While embodiments of the present invention have been shown and described, it will be appreciated by those skilled in the art that various changes, modifications, substitutions, and variations may be made to these embodiments without departing from the principles and spirit of the invention, and that the scope of the invention is defined by the appended claims and their equivalents.
Claims
1. A method for extracting active polypeptides from freshwater fish, characterized in that: The following steps are involved: (1) Take fresh freshwater fish bones, wash them and homogenize them; (2) adding deionized water containing mandelic acid to the homogenate at a solid-liquid ratio of 3-5:1, wherein the deionized water contains 0.1-0.3 wt % of a mixture of D-mandelic acid and L-mandelic acid, and the weight ratio of D-mandelic acid to L-mandelic acid is 52-58:0.3-0.7; (3) The mixture was stirred at a constant temperature of 47-52°C, the pH was adjusted to 6.7-7.0, and the complex enzyme preparation was added and enzymatically hydrolyzed for 4-6 hours; (4) Nitrogen was introduced every 20-30 min during the enzymatic hydrolysis process at a rate of 15-25 g / h; (5) After the enzymatic hydrolysis is completed, the enzymatic hydrolyzate is heated to 92-95°C and maintained for 8-12 minutes to inactivate the enzyme, the pH is adjusted to neutral, and then centrifuged at 4-10°C and 7000-8000 r / min for 10-15 minutes. The supernatant is collected to obtain the active polypeptide.
2. The method for extracting active polypeptides from freshwater fish according to claim 1, wherein: The complex enzyme preparation consists of 4-10 parts of neutral protease, 3-5 parts of papain and 1-2 parts of trypsin.
3. The method for extracting active polypeptides from freshwater fish according to claim 1, wherein: The active polypeptide has a molecular weight of 2000-3000 Da and contains aspartic acid, threonine, glutamic acid, glycine, alanine, isoleucine, tyrosine, phenylalanine, histidine, lysine and arginine.