Preparation method of high-activity sea cucumber peptide

Abstract

The invention relates to the technical field of sea cucumber peptide extraction, and particularly discloses a preparation method of high-activity sea cucumber peptide. The preparation method of the high-activity sea cucumber peptide provided by the invention comprises the following steps: raw material pretreatment, directional enzymolysis, refining concentration and drying. The pretreatment step comprises the following steps: carrying out ultrasonic treatment on the sea cucumber homogenate by adopting a composite pretreatment agent, and then centrifuging to obtain a sea cucumber pretreatment solution; wherein 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 8-10 wt% of a chitin suspension and 0.8-1.2 wt% of an ergothioneine aqueous solution in a volume ratio of 1: (2-3). According to the preparation method of the sea cucumber peptide, the sea cucumber peptide which is high in yield, high in activity, low in heavy metal residue and free of special fishy smell can be obtained, and large-scale production of the sea cucumber peptide can be achieved.

Description

TECHNICAL FIELD

[0001] The application relates to a preparation method of high-activity sea cucumber peptide. BACKGROUND

[0002] As a marine bioactive component rich in various amino acids and functional sequences, sea cucumber peptide has multiple effects such as antioxidation, tissue repair and immunoregulation, and has broad application prospects in the fields of food, cosmetics and medicine.

[0003] However, the existing sea cucumber peptide preparation technology still has various defects, which seriously restrict the industrialization and high-value application of sea cucumber peptide. First, the enzymolysis efficiency is low, resulting in low peptide yield and resource waste; second, in the sea cucumber peptide extraction and processing process, heat-sensitive functional peptide segments are prone to degradation, and the peptide activity retention rate is low; third, sea cucumber peptide is prone to have problems such as heavy metal residue, characteristic fishy smell and color, which limits the wide application of sea cucumber peptide products.

[0004] Therefore, it is of great significance to develop a high-efficiency, high-quality and low-cost sea cucumber peptide preparation method. SUMMARY

[0005] In order to overcome the problems of low extraction efficiency and low peptide activity retention rate existing in the existing sea cucumber peptide preparation method, the application provides a preparation method of high-activity sea cucumber peptide.

[0006] In a first aspect, the application provides a preparation method of high-activity sea cucumber peptide, which adopts the following technical scheme: A preparation method of high-activity sea cucumber peptide, comprising the following steps: raw material pretreatment, directional enzymolysis, refining and concentration and drying; The pretreatment step is: the sea cucumber homogenate is subjected to ultrasonic treatment by using a composite pretreatment agent, and then centrifuged to obtain a sea cucumber pretreatment liquid; The volume ratio of the sea cucumber homogenate and the composite pretreatment agent is 1: (1.5-3); the composite pretreatment agent is a mixture of 8-10 wt% chitin suspension and 0.8-1.2 wt% ergothioneine aqueous solution with a volume ratio of 1: (2-3).

[0007] The application provides a preparation method of high-activity sea cucumber peptide, wherein the pretreatment step adopts a specific ratio of chitin suspension and ergothioneine aqueous solution to perform synergistic ultrasonic treatment on the sea cucumber homogenate, which can fully dissolve the proteins in the sea cucumber tissue and maintain the activity of the peptide components and the purity of the system, laying a foundation for efficient development of subsequent processes such as enzymolysis, and finally obtaining sea cucumber peptide with high yield, high activity, low heavy metal residue and no special fishy smell.

[0008] The application adopts chitin as a pretreatment agent, which can physically pierce the sea cucumber tissue fibers, break its dense structure, accelerate the full dissolution of internal proteins, and also adsorb heavy metal ions such as cadmium and lead, and fishy smell precursor substances such as trimethylamine and dimethylamine, thereby removing impurities from the source, reducing the processing pressure of subsequent refining and concentration processes, and improving the purity of the pretreatment liquid. Ergothioneine can efficiently remove active oxygen free radicals generated in the ultrasonic treatment process, avoid the structural degradation of heat-sensitive active ingredients such as RGD peptide due to oxidation, and maximize the biological activity of sea cucumber peptide. On the other hand, its natural molecular structure is stable and will not have an antagonistic reaction with enzymes in the subsequent directional enzymolysis process, will not interfere with the specific cleavage of the peptide bond by protease, and will ensure that the enzymolysis reaction proceeds efficiently and orderly.

[0009] Optionally, the power of the ultrasonic treatment is 280-350W, the frequency is 30-40kHz, the temperature is 15-22℃, and the time is 80-140min.

[0010] Optionally, the directional enzymolysis step uses a complex enzyme preparation to enzymolyze the sea cucumber pretreatment liquid, the temperature is 40-45℃, and the time is 2.5-3h. The complex enzyme preparation contains alginate lyase and ficin.

[0011] In the application, alginate lyase can specifically degrade the mucopolysaccharide matrix such as fucoidan and alginate that wraps the protein in the sea cucumber tissue, break the dense tissue barrier, and open the protein dissolution channel, making it easier for ficin to contact the core protein substrate. At the same time, its mild degradation characteristics will not damage the protein main structure, avoiding the loss of active peptide precursors due to improper matrix fragmentation. Ficin, as a plant-derived cysteine protease, can cleave the peptide bonds at the ends of aromatic and basic amino acids in protein molecules, degrading large molecular proteins into small molecular peptides with a molecular weight of ≤2000Da. By using the above two enzymes in a specific ratio, the application can not only reduce the amount of enzyme preparation and improve the efficiency of enzymolysis, but also avoid excessive degradation of active peptides and ensure the activity of peptides, effectively solving the problem that traditional enzyme preparations cannot balance enzymolysis efficiency and peptide activity.

[0012] Optionally, the weight ratio of alginate lyase to ficin is (0.2-0.8):1.

[0013] Optionally, the amount of the complex enzyme preparation added is 0.9-1.2wt% of the sea cucumber pretreatment liquid.

[0014] Optionally, in the directional enzymolysis step, 0.05% cysteine is also added to the sea cucumber pretreatment liquid.

[0015] Optionally, the refining and concentrating step is: adding activated carbon to the inactivated enzymatic hydrolysate, stirring at 50-60℃ for 20-40min, and filtering; then sequentially using ultrafiltration membrane and nanofiltration membrane for dialysis and concentration to obtain sea cucumber peptide concentrate. Optionally, the drying temperature is 95-115℃.

[0016] In the second aspect, the application provides a high-activity sea cucumber peptide obtained by the above method.

[0017] In summary, the application has the following advantages: 1. The application provides a preparation method of high-activity sea cucumber peptide, which uses a mixture of 8-10wt% chitin suspension and 0.8-1.2wt% ergothioneine aqueous solution with a volume ratio of 1:(2-3) as a composite pretreatment agent, and first mixes sea cucumber homogenate and the composite pretreatment agent with a volume ratio of 1:(1.5-3) for ultrasonic treatment, and then sequentially performs directional enzymolysis, refining and concentration, and drying, so as to realize efficient extraction of peptides from sea cucumber, and obtain high-activity, low heavy metal residue, and no special fishy smell sea cucumber peptide.

[0018] 2. The application uses a mixture of 0.2-0.8:1 of alginate lyase and ficin as a composite enzyme preparation, which can improve the efficiency of enzymolysis, avoid excessive degradation of active peptides, and ensure the activity of peptides, effectively solving the problem that the traditional enzyme preparation cannot balance the enzymolysis efficiency and peptide activity. BRIEF DESCRIPTION OF DRAWINGS

[0019] Figure 1 The application provides a preparation method of high-activity sea cucumber peptide. DETAILED DESCRIPTION

[0020] The application provides a preparation method of high-activity sea cucumber peptide, which includes the following steps: (1) Raw material pretreatment: removing the internal organs of fresh sea cucumber, crushing the sea cucumber wall after washing, and then adding water to prepare 25-40wt% sea cucumber homogenate; adding a composite pretreatment agent to the sea cucumber homogenate, and ultrasonic treating under the conditions of a power of 280-350W, a frequency of 30-40kHz, and a temperature of 15-22℃ for 80-140min, and then standing, and then centrifuging at 5000-7000r / min for 5-10min to remove the precipitate, to obtain sea cucumber pretreatment liquid; The composite pretreatment agent is a mixture of 8-10wt% chitin suspension and 0.8-1.2wt% ergothioneine aqueous solution with a volume ratio of 1:(2-3); the volume ratio of sea cucumber homogenate and composite pretreatment agent is 1:(1.5-3).

[0021] (2) Directional enzymolysis: adjust the pH of the sea cucumber pretreatment liquid to 6.8-7.3, and then add a composite enzyme preparation, the addition amount of the composite enzyme preparation being 0.9-1.2wt% of the sea cucumber pretreatment liquid, and constant-temperature enzymolysis is carried out for 2.5-3h, when the degree of hydrolysis reaches 30-40%, the temperature is raised to 80-90℃ for 15-25min to inactivate the enzyme, and then the temperature is cooled to room temperature to obtain an enzymolysis liquid; The composite enzyme preparation comprises alginate lyase and ficin in a weight ratio of (0.2-0.8):1.

[0022] (3) Concentration: add activated carbon to the inactivated enzymolysis liquid, stir at 50-60℃ for 20-40min, and then filter; and then dialyze and concentrate through an ultrafiltration membrane and a nanofiltration membrane to obtain a sea cucumber peptide concentrate with a solid content of 25-35%; (4) Drying: spray dry the sea cucumber peptide concentrate at a temperature of 95-115℃, crush, and pass through an 80-120 mesh sieve to obtain a high-activity sea cucumber peptide.

[0023] In the present application, chitin is purchased from Zhengzhou Jiuting Chemical Product Co., Ltd.; ergothioneine is purchased from Shanghai Zhongfeng Biological Technology Co., Ltd.; alginate lyase is purchased from Jiangsu Ruide Biological Engineering Co., Ltd.; the enzyme activity of the alginate lyase is ≥500U / g, and the enzyme activity of the ficin is ≥1000U / g; the raw materials, reagents, solvents and the like used in the present application can be obtained by commercial purchase.

[0024] The present application will be further described in detail in combination with the following embodiment, performance detection test and drawing description. Examples 1-6

[0025] Examples 1-6 respectively provide a preparation method of a high-activity sea cucumber peptide.

[0026] The differences between the above examples are as follows: the addition amount of the composite pretreatment agent and the volume ratio of the chitin suspension and the ergothioneine aqueous solution in the composite pretreatment agent, and the specific conditions are shown in Table 1.

[0027] The preparation method of the high-activity sea cucumber peptide provided in Examples 1-6 comprises the following steps: (1) Raw material pretreatment: remove the internal organs and sundries of fresh sea cucumber, crush the sea cucumber wall after washing, and then add water to prepare a 30wt% sea cucumber homogenate liquid; add a composite pretreatment agent to 100mL of the sea cucumber homogenate liquid according to Table 1, and ultrasonically treat under the conditions of a power of 300W, a frequency of 40kHz and a temperature of 19℃ for 120min, stand, and then centrifuge at 6000r / min for 10min to remove the precipitate to obtain a sea cucumber pretreatment liquid.

[0028] (2) Directional enzymolysis: adjust the pH of the sea cucumber pretreatment liquid to 7.0, and then heat to 42℃, then according to the amount of sea cucumber pretreatment liquid, add 1wt% composite enzyme preparation (weight ratio of 0.6:1 of alginate lyase and ficin) and 0.05% cysteine, constant temperature stirring enzymolysis for 3h, when the degree of hydrolysis reaches 35%, heat to 90℃ for 20min to inactivate the enzyme, cool to room temperature to obtain the enzyme hydrolysate.

[0029] (3) Concentration: add activated carbon to the inactivated enzyme hydrolysate, stir at 50℃ for 30min, filter; then dialyze and concentrate through ultrafiltration membrane and nanofiltration membrane in turn, to obtain sea cucumber peptide concentrate with a solid content of 30%; (4) Drying; spray dry the sea cucumber peptide concentrate at a temperature of 110℃, crush, pass through a 120 mesh sieve, and obtain high-activity sea cucumber peptide.

[0030] Table 1: The amount of composite pretreatment agent and its component ratio in examples 1-6 Examples 7-10

[0031] Examples 7-10 respectively provide a preparation method of high-activity sea cucumber peptide.

[0032] The above examples are different from example 2 in that the component ratio of the composite enzyme preparation is as shown in the following table 2.

[0033] Table 2: The component ratio of the composite enzyme preparation in example 2, examples 7-10 Comparative example 1

[0034] Comparative example 1 provides a preparation method of high-activity sea cucumber peptide.

[0035] The above comparative example is different from example 2 in that the amount of composite pretreatment agent is 100mL, i.e. the volume ratio of sea cucumber homogenate liquid to composite pretreatment agent is 1:1. Comparative example 2

[0036] Comparative example 2 provides a preparation method of high-activity sea cucumber peptide.

[0037] The above comparative example is different from example 2 in that the composite pretreatment agent is replaced by an equal amount of 10wt% chitin suspension. Comparative example 3

[0038] Comparative example 3 provides a preparation method of high-activity sea cucumber peptide.

[0039] The above comparative example is different from example 2 in that the composite pretreatment agent is replaced by an equal amount of 1wt% ergothioneine aqueous solution. Comparative Example 4

[0040] Comparative Example 4 provides a preparation method of a high-activity sea cucumber peptide.

[0041] The above comparative examples and Example 2 differ in that the composite pretreatment agent is a mixture of 8-10 wt% chitin suspension and 0.8-1.2 wt% aqueous ergothioneine in a volume ratio of 1:1. Performance detection test

[0042] 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, and the protein content in the raw sea cucumber, the peptide content and the total peptide amount in the product sea cucumber peptide were detected, and the peptide yield was calculated, and the results are shown in Table 3. The calculation formula of the peptide yield is as follows: Peptide yield = (total peptide amount in sea cucumber peptide / total protein mass in sea cucumber) x 100% 2. Activity detection: according to the method of GB / T 31740.2-2015, the DPPH free radical scavenging rate of the active peptide was detected to evaluate the activity of the active peptide; the higher the DPPH free radical scavenging rate, the stronger the activity of the sea cucumber peptide.

[0043] 3. Heavy metal content: the cadmium and lead contents in the sea cucumber peptide were detected according to GB 5009.12-2017 and GB 5009.15-2014, respectively.

[0044] Table 3: Detection results of sea cucumber peptides obtained in Examples 1-10 and Comparative Examples 1-4

[0045] According to the detection results in Table 3, the peptide yield obtained by the preparation method provided in Examples 1-10 is 85.8-95.1% (≥85%), the DPPH free radical scavenging rate is 84.2-91.3% (≥80%), the cadmium content is ≤0.02 mg / kg, and the lead content is ≤0.03 mg / kg; while the peptide yield obtained by the preparation method provided in Comparative Examples 1-4 is 71.4-89.0%, the DPPH free radical scavenging rate is 72.6-80.4%, and the cadmium content in the sea cucumber peptide obtained in Comparative Example 3 is 0.027 mg / kg, and the lead content is 0.033 mg / kg. Therefore, it is shown that the present application uses a mixture of 8-10 wt% chitin suspension and 0.8-1.2 wt% aqueous ergothioneine 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 realize efficient extraction of peptides from sea cucumber, and obtain sea cucumber peptides with high activity, low heavy metal residue, and no special fishy smell.

[0046] 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).

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, The targeted enzymatic hydrolysis step involves enzymatically hydrolyzing the sea cucumber pretreatment solution with a compound enzyme preparation at a temperature of 40-45℃ for 2.5-3 hours. The compound enzyme preparation contains alginate lyase and fig protease.

4. The method for preparing highly active sea cucumber peptides according to claim 3, characterized in that, The weight ratio of alginate lyase to fig protease is (0.2-0.8):

1.

5. The method for preparing highly active sea cucumber peptides according to claim 3 or 4, characterized in that, The amount of the compound enzyme preparation added is 0.9-1.2 wt% of the sea cucumber pretreatment solution.

6. The method for preparing highly active sea cucumber peptides according to claim 1, characterized in that, In the directional enzymatic hydrolysis step, 0.05% cysteine ​​was also added to the sea cucumber pretreatment solution.

7. 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.

8. The method for preparing highly active sea cucumber peptides according to claim 1, characterized in that, The drying temperature is 95-115℃.

9. 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 1-8.

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