Preparation method of high-activity sea cucumber peptide
By using a composite pretreatment agent and targeted enzymatic hydrolysis technology, the problems of low enzymatic hydrolysis efficiency and low activity retention rate in the preparation of sea cucumber peptides have been solved, resulting in sea cucumber peptides with high activity, low heavy metal residue, and no fishy smell, thus promoting the industrial application of sea cucumber peptides.
Patent Information
- Authority / Receiving Office
- CN · China
- Patent Type
- Patents(China)
- Current Assignee / Owner
- QINGDAO SUNWAY FOOD
- Filing Date
- 2026-02-11
- Publication Date
- 2026-06-02
AI Technical Summary
Existing sea cucumber peptide preparation technologies suffer from low enzymatic hydrolysis efficiency, low peptide activity retention, heavy metal residues, and fishy odor, which limit their industrial application.
The sea cucumber homogenate was ultrasonically treated with a composite pretreatment agent (8-10wt% chitin suspension and 0.8-1.2wt% ergothioneine aqueous solution), followed by targeted enzymatic hydrolysis with alginate lyase and fig protease. Then, it was purified, concentrated and dried to remove impurities and retain activity.
It achieves efficient extraction of highly active sea cucumber peptides with low heavy metal residue and no fishy smell, improving enzymatic hydrolysis efficiency and peptide activity retention rate.
Smart Images
Figure CN121718601B_ABST
Abstract
Description
Technical Field
[0001] This application relates to the field of sea cucumber peptide extraction technology, specifically to a method for preparing highly active sea cucumber peptides. Background Technology
[0002] Sea cucumber peptides, as marine bioactive components rich in various amino acids and functional sequences, have multiple effects such as anti-oxidation, tissue repair, and immune regulation, and have broad application prospects in the fields of food, cosmetics, and medicine.
[0003] However, existing sea cucumber peptide preparation technologies still have several shortcomings that severely restrict the industrialization and high-value application of sea cucumber peptides. First, low enzymatic hydrolysis efficiency leads to low peptide yields and resource waste. Second, during the extraction and processing of sea cucumber peptides, heat-sensitive functional peptides are easily degraded, resulting in low peptide activity retention. Third, sea cucumber peptides are prone to problems such as heavy metal residues, characteristic fishy odor, and discoloration, limiting the widespread application of sea cucumber peptide products.
[0004] Therefore, developing an efficient, high-quality, and low-cost method for preparing sea cucumber peptides is of great significance. Summary of the Invention
[0005] In order to overcome the problems of low extraction efficiency and low peptide activity retention in existing sea cucumber peptide preparation methods, this application provides a method for preparing highly active sea cucumber peptides.
[0006] In a first aspect, this application provides a method for preparing highly active sea cucumber peptides, employing the following technical solution:
[0007] A method for preparing highly active sea cucumber peptides includes the following steps: raw material pretreatment, targeted enzymatic hydrolysis, purification and concentration, and drying;
[0008] The pretreatment step is as follows: the sea cucumber homogenate is ultrasonically treated with a composite pretreatment agent and then centrifuged to obtain the sea cucumber pretreatment solution.
[0009] The volume ratio of the sea cucumber homogenate to the composite pretreatment agent is 1:(1.5-3); the composite pretreatment agent is a mixture of 8-10wt% chitin suspension and 0.8-1.2wt% ergothioneine aqueous solution in a volume ratio of 1:(2-3).
[0010] This application provides a method for preparing highly active sea cucumber peptides. The pretreatment step involves synergistic ultrasonic treatment of sea cucumber homogenate with a chitin suspension and ergothioneine aqueous solution in a specific ratio. This method can fully dissolve the proteins in the sea cucumber tissue while maintaining the activity of the peptide components and the purity of the system. This lays the foundation for the efficient implementation of subsequent processes such as enzymatic hydrolysis, and ultimately yields sea cucumber peptides with high yield, high activity, low heavy metal residue, and no special fishy smell.
[0011] This application uses chitin as a pretreatment agent. On one hand, it can physically puncture the fibers of sea cucumber tissue, breaking its dense structure and accelerating the full dissolution of internal proteins. On the other hand, it can adsorb heavy metal ions such as cadmium and lead, as well as odor precursors such as trimethylamine and dimethylamine, removing impurities at the source, reducing the processing pressure of subsequent refining and concentration processes, and improving the purity of the pretreatment solution. Ergothionein can efficiently remove reactive oxygen free radicals generated during ultrasonic treatment, preventing the structural degradation of heat-sensitive active ingredients such as RGD peptides due to oxidation, thus maximizing the preservation of the bioactivity of sea cucumber peptides. Furthermore, its natural molecular structure is stable and will not antagonize enzymes in subsequent targeted enzymatic hydrolysis processes, nor interfere with the specific cleavage of peptide bonds by proteases, ensuring the efficient and orderly conduct of the enzymatic hydrolysis reaction.
[0012] Optionally, the ultrasonic treatment has a power of 280-350W, a frequency of 30-40kHz, a temperature of 15-22℃, and a duration of 80-140min.
[0013] Optionally, the directional enzymatic hydrolysis step uses a compound enzyme preparation to enzymatically hydrolyze the sea cucumber pretreatment solution at a temperature of 40-45℃ for 2.5-3 hours.
[0014] The compound enzyme preparation contains alginate lyase and fig protease.
[0015] In this application, alginate lyase can specifically degrade the fucoidan, alginate, and other mucopolysaccharide matrices that encapsulate proteins in sea cucumber tissues, breaking down the dense tissue barrier and opening protein dissolution channels, facilitating easier access for fig protease to the core protein substrate. Simultaneously, its mild degradation characteristics do not damage the protein's main structure, avoiding the loss of bioactive peptide precursors due to improper matrix fragmentation. Fig protease, as a plant-derived cysteine protease, can cleave the terminal peptide bonds of aromatic and basic amino acids in protein molecules, degrading large proteins into small peptides with a molecular weight ≤2000 Da. This application utilizes the two enzymes in a specific ratio, which reduces enzyme dosage, improves enzymatic hydrolysis efficiency, and prevents excessive degradation of bioactive peptides, ensuring peptide activity. This effectively solves the problem of traditional enzyme preparations where enzymatic hydrolysis efficiency and peptide activity are difficult to balance.
[0016] Optionally, the weight ratio of the alginate lyase to the fig protease is (0.2-0.8):1.
[0017] Optionally, the amount of the compound enzyme preparation added is 0.9-1.2 wt% of the sea cucumber pretreatment solution.
[0018] Optionally, in the directional enzymatic hydrolysis step, 0.05% cysteine is added to the sea cucumber pretreatment solution.
[0019] Optionally, the purification and concentration step is as follows: add activated carbon to the inactivated enzyme hydrolysate, stir at 50-60℃ for 20-40 min, and filter; then concentrate by dialysis using an ultrafiltration membrane and a nanofiltration membrane in sequence to obtain a concentrated sea cucumber peptide solution;
[0020] Optionally, the drying temperature is 95-115°C.
[0021] Secondly, this application provides a highly active sea cucumber peptide, which is obtained using the aforementioned method for preparing highly active sea cucumber peptides.
[0022] In summary, this application has the following beneficial effects:
[0023] 1. This application provides a method for preparing highly active sea cucumber peptides. The method uses a mixture of 8-10wt% chitin suspension and 0.8-1.2wt% ergothioneine aqueous solution at a volume ratio of 1:(2-3) as a composite pretreatment agent. First, the sea cucumber homogenate and the composite pretreatment agent are mixed and ultrasonically mixed at a volume ratio of 1:(1.5-3). Then, the mixture is subjected to directional enzymatic hydrolysis, purification and concentration, and drying in sequence. This method can achieve efficient extraction of peptides from sea cucumbers and obtain highly active sea cucumber peptides with low heavy metal residue and no special fishy smell.
[0024] 2. This application uses alginate lyase and fig protease in a weight ratio of (0.2-0.8):1 as a compound enzyme preparation, which can improve the enzymatic hydrolysis efficiency on the one hand, and avoid excessive degradation of active peptides and ensure peptide activity on the other hand, effectively solving the problem that traditional enzyme preparations cannot simultaneously achieve both enzymatic hydrolysis efficiency and peptide activity. Attached Figure Description
[0025] Figure 1 This is a flowchart of the preparation method of the highly active sea cucumber peptide provided in this application. Detailed Implementation
[0026] This application provides a method for preparing highly active sea cucumber peptides, comprising the following steps:
[0027] (1) Raw material pretreatment: Remove the internal organs and impurities from fresh sea cucumbers, clean the sea cucumber walls and crush them, then add water to make a sea cucumber homogenate of 25-40wt%; add a compound pretreatment agent to the sea cucumber homogenate, and ultrasonically treat it for 80-140min at a power of 280-350W, a frequency of 30-40kHz and a temperature of 15-22℃, let it stand, and then centrifuge it at 5000-7000r / min for 5-10min to remove the precipitate and obtain the sea cucumber pretreatment solution;
[0028] The composite pretreatment agent is a mixture of 8-10wt% chitin suspension and 0.8-1.2wt% ergothioneine aqueous solution in a volume ratio of 1:(2-3); the volume ratio of sea cucumber homogenate to composite pretreatment agent is 1:(1.5-3).
[0029] (2) Targeted enzymatic hydrolysis: Adjust the pH of the sea cucumber pretreatment solution to 6.8-7.3, raise the temperature to 40-45℃, and then add the compound enzyme preparation. The amount of compound enzyme preparation added is 0.9-1.2wt% of the sea cucumber pretreatment solution. Stir at a constant temperature for 2.5-3h for enzymatic hydrolysis. When the degree of hydrolysis reaches 30-40%, raise the temperature to 80-90℃ for 15-25min to inactivate the enzyme, and cool to room temperature to obtain the enzymatic hydrolysate.
[0030] The compound enzyme preparation contains alginate lyase and fig protease in a weight ratio of (0.2-0.8):1.
[0031] (3) Refining and concentration: Add activated carbon to the inactivated enzyme hydrolysate, stir at 50-60℃ for 20-40 min, and filter; then concentrate by dialysis through ultrafiltration membrane and nanofiltration membrane to obtain sea cucumber peptide concentrate with a solid content of 25-35%;
[0032] (4) Drying: The sea cucumber peptide concentrate is spray-dried at a temperature of 95-115℃, pulverized, and passed through an 80-120 mesh sieve to obtain highly active sea cucumber peptides.
[0033] In this application, chitin was purchased from Zhengzhou Jiuting Chemical Products Co., Ltd.; ergothioneine was purchased from Shanghai Zhongfeng Biotechnology Co., Ltd.; alginate lyase was purchased from Jiangsu Ruiduo Bioengineering Co., Ltd.; the enzyme activity of alginate lyase was ≥500U / g, and the enzyme activity of fig protease was ≥1000U / g; the raw materials, reagents, solvents and other materials used in this application can all be obtained commercially.
[0034] The present application will be further described in detail below with reference to embodiments, performance testing tests and accompanying drawings.
[0035] Examples 1-6
[0036] Examples 1-6 provide a method for preparing highly active sea cucumber peptides.
[0037] The difference in the above embodiments lies in the amount of composite pretreatment agent added and the volume ratio of chitin suspension and ergothioneine aqueous solution in the composite pretreatment agent, as shown in Table 1 below.
[0038] The preparation method of highly active sea cucumber peptides provided in Examples 1-6 includes the following steps:
[0039] (1) Raw material pretreatment: Remove the internal organs and impurities from fresh sea cucumbers, clean the sea cucumber walls and crush them, then add water to make a 30wt% sea cucumber homogenate; add the compound pretreatment agent to 100mL of sea cucumber homogenate as shown in Table 1, and sonicate for 120min at a power of 300W, a frequency of 40kHz and a temperature of 19℃, let stand, and then centrifuge at 6000r / min for 10min to remove the precipitate to obtain the sea cucumber pretreatment solution.
[0040] (2) Targeted enzymatic hydrolysis: Adjust the pH of the sea cucumber pretreatment solution to 7.0, raise the temperature to 42℃, and then add 1wt% of compound enzyme preparation (alginic acid lyase and fig protease with a weight ratio of 0.6:1) and 0.05% cysteine according to the amount of sea cucumber pretreatment solution. Stir at a constant temperature for 3 hours for enzymatic hydrolysis. When the degree of hydrolysis reaches 35%, raise the temperature to 90℃ for 20 minutes to inactivate the enzyme, and cool to room temperature to obtain the enzymatic hydrolysate.
[0041] (3) Refining and concentration: Add activated carbon to the inactivated enzyme hydrolysate, stir at 50°C for 30 min, and filter; then concentrate by dialysis through ultrafiltration membrane and nanofiltration membrane to obtain sea cucumber peptide concentrate with a solid content of 30%;
[0042] (4) Drying: The sea cucumber peptide concentrate is spray-dried at 110°C, pulverized, and passed through a 120-mesh sieve to obtain highly active sea cucumber peptide.
[0043] Table 1. Amount and composition ratio of the composite pretreatment agent in Examples 1-6
[0044]
[0045] Examples 7-10
[0046] Examples 7-10 provide a method for preparing highly active sea cucumber peptides.
[0047] The difference between the above embodiments and Embodiment 2 is that the component ratio of the compound enzyme preparation is shown in Table 2 below.
[0048] Table 2. Composition ratios of the compound enzyme preparations in Examples 2 and 7-10
[0049] Comparative Example 1
[0050] Comparative Example 1 provides a method for preparing highly active sea cucumber peptides.
[0051] The difference between the above comparative example and Example 2 is that the amount of composite pretreatment agent added is 100 mL, that is, the volume ratio of sea cucumber homogenate to composite pretreatment agent is 1:1. Comparative Example 2
[0052] Comparative Example 2 provides a method for preparing highly active sea cucumber peptides.
[0053] The difference between the above comparative example and Example 2 is that the composite pretreatment agent was replaced with an equal amount of 10wt% chitosan suspension. Comparative Example 3
[0054] Comparative Example 3 provides a method for preparing highly active sea cucumber peptides.
[0055] The difference between the above comparative example and Example 2 is that the composite pretreatment agent was replaced with an equal amount of 1 wt% ergothioneine aqueous solution. Comparative Example 4
[0056] Comparative Example 4 provides a method for preparing highly active sea cucumber peptides.
[0057] The difference between the above comparative example and Example 2 is that the composite pretreatment agent is a mixture of 8-10wt% chitin suspension and 0.8-1.2wt% ergothioneine aqueous solution in a volume ratio of 1:1.
[0058] Performance testing
[0059] 1. Purity and Yield Detection: Sea cucumber peptides were prepared according to the methods provided in Examples 1-10 and Comparative Examples 1-4, respectively. The protein content in the raw sea cucumber, the peptide content in the product sea cucumber peptides, and the total peptide content were detected, and the peptide yield was calculated. The results are shown in Table 3. The formula for calculating the peptide yield is as follows:
[0060] Peptide yield = (Total peptides in sea cucumber / Total protein in sea cucumber) × 100%
[0061] 2. Activity detection: The activity of the active peptide was evaluated by detecting the scavenging rate of DPPH free radicals by the active peptide according to the method of GB / T 31740.2-2015; the higher the DPPH free radical scavenging rate, the stronger the activity of the sea cucumber peptide.
[0062] 3. Heavy metal content: The cadmium and lead content in sea cucumber peptides were tested according to GB 5009.12-2017 and GB 5009.15-2014, respectively.
[0063] Table 3. Detection results of sea cucumber peptides obtained in Examples 1-10 and Comparative Examples 1-4
[0064]
[0065] According to the test results in Table 3, the peptide yield obtained by the preparation methods provided in Examples 1-10 was 85.8-95.1% (≥85%), the DPPH free radical scavenging rate was 84.2-91.3% (≥80%), the cadmium content was ≤0.02 mg / kg, and the lead content was ≤0.03 mg / kg; while the peptide yield obtained by the preparation methods provided in Comparative Examples 1-4 was 71.4-89.0%, and the DPPH free radical scavenging rate was 72.6-80.4%; among them, the cadmium content in the sea cucumber peptide obtained in Comparative Example 3 was 0.027 mg / kg, and the lead content was 0.033 mg / kg. Therefore, this application uses a mixture of 8-10wt% chitin suspension and 0.8-1.2wt% ergothioneine aqueous solution in a volume ratio of 1:(2-3) as a composite pretreatment agent, and controls the volume ratio of sea cucumber homogenate and composite pretreatment agent in the range of 1:(1.5-3), which can achieve efficient extraction of peptides from sea cucumbers and obtain sea cucumber peptides with high activity, low heavy metal residue and no special fishy smell.
[0066] Although the present invention has been described in detail above with general descriptions and specific embodiments, modifications or improvements can be made to it, which will be obvious to those skilled in the art. Therefore, all such modifications or improvements made without departing from the spirit of the present invention fall within the scope of protection claimed by the present invention.
Claims
1. A method for preparing highly active sea cucumber peptides, characterized in that, Includes the following steps: Raw material pretreatment, targeted enzymatic hydrolysis, refining and concentration, and drying; The pretreatment step is as follows: the sea cucumber homogenate is ultrasonically treated with a composite pretreatment agent and then centrifuged to obtain the sea cucumber pretreatment solution. The volume ratio of the sea cucumber homogenate to the composite pretreatment agent is 1:(1.5-3); the composite pretreatment agent is a mixture of 8-10wt% chitin suspension and 0.8-1.2wt% ergothioneine aqueous solution in a volume ratio of 1:(2-3). The targeted enzymatic hydrolysis step is as follows: the sea cucumber pretreatment solution is enzymatically hydrolyzed using a compound enzyme preparation containing alginate lyase and fig protease in a weight ratio of (0.2-0.8):1 and 0.05% cysteine.
2. The method for preparing highly active sea cucumber peptides according to claim 1, characterized in that, The ultrasonic treatment has a power of 280-350W, a frequency of 30-40kHz, a temperature of 15-22℃, and a duration of 80-140min.
3. The method for preparing highly active sea cucumber peptides according to claim 1, characterized in that, In the directional enzymatic hydrolysis step, the enzymatic hydrolysis temperature is 40-45℃ and the time is 2.5-3h.
4. The method for preparing highly active sea cucumber peptides according to claim 1, characterized in that, The amount of the compound enzyme preparation added is 0.9-1.2 wt% of the sea cucumber pretreatment solution.
5. The method for preparing highly active sea cucumber peptides according to claim 1, characterized in that, The purification and concentration steps are as follows: add activated carbon to the inactivated enzyme hydrolysate, stir at 50-60℃ for 20-40 minutes, and filter; then concentrate by dialysis using ultrafiltration membrane and nanofiltration membrane in sequence to obtain sea cucumber peptide concentrate.
6. The method for preparing highly active sea cucumber peptides according to claim 1, characterized in that, The drying temperature is 95-115℃.
7. A highly active sea cucumber peptide, characterized in that, The highly active sea cucumber peptide was obtained using the preparation method described in any one of claims 1-6.