Preparation method of phascolosoma esculenta protein peptide and application thereof in cell repair and whitening

Abstract

The application provides a preparation method of a protein peptide of Phascolosoma esculenta and application of the protein peptide in cell repair and whitening. The preparation method of the protein peptide of Phascolosa esculenta comprises the following steps: cutting fresh and live Phascolosoma esculenta after cleaning, homogenizing the Phascolosoma esculenta by adding water, adding flavor protease and neutral protease, carrying out enzymolysis for 6 h under the condition of 55 DEG C, carrying out enzyme inactivation treatment by heating at 100 DEG C for 15 min, centrifuging to take supernatant, ultrafiltration, and freeze-drying, so that the protein peptide of Phascolosoma esculenta is prepared. The protein peptide of Phascolosoma esculenta can promote cell repair and inhibit melanin synthesis of zebra fish embryos.

Description

TECHNICAL FIELD

[0001] The present application relates to the fields of food technology and medicine, in particular to a preparation method of Phascolosoma esculenta protein peptide and its application in cell repair and whitening. BACKGROUND

[0002] Phascolosoma esculenta is a kind of marine annelid, commonly known as soil bamboo shoot, belonging to the phylum Sipuncula, class Sipunculida, order Sipunculida and family Sipunculidae. Phascolosoma esculenta mainly inhabits in the environment of shallow sand and mud, and is widely distributed in Fujian, Guangdong and Guangxi of China. Phascolosoma esculenta is rich in protein, unsaturated fatty acids, polysaccharides, trace elements and other nutrients, and is a typical high-protein and low-fat marine product.

[0003] Research reports show that Phascolosoma esculenta protein peptide generated by protease hydrolysis of Phascolosoma esculenta has the effects of promoting wound healing, antibacterial, antioxidant, antihypertensive, antitumor and immunomodulation. In addition, Phascolosoma esculenta protein peptide has a lower molecular weight and is more easily absorbed by the human body, and has the ability to be absorbed through the epidermis. Based on the above advantages, Phascolosoma esculenta protein peptide has attracted the attention of consumers, manufacturers and researchers in the fields of food, medicine and cosmetics, and is considered as an active substance with wide application prospect. However, there is no related research report on the whitening effect evaluation of Phascolosoma esculenta protein peptide by zebrafish test, which cannot provide theoretical basis support for the whitening effect of Phascolosoma esculenta protein peptide. Therefore, the application of Phascolosoma esculenta protein peptide in whitening and promoting wound healing will become a new focus in the field of wound repair and whitening, and has good application prospect. SUMMARY

[0004] The purpose of the present application is to provide a preparation method of Phascolosoma esculenta protein peptide and its application in cell repair and whitening.

[0005] To achieve the above purpose, the technical scheme adopted by the present application is as follows:

[0006] A preparation method of Phascolosoma esculenta protein peptide, comprising the following steps:

[0007] S1: taking fresh Phascolosoma esculenta as raw material, washing it with water, draining the water, cutting it, adding 6 times the mass of water of the raw material, and homogenizing it into Phascolosoma esculenta homogenate by a homogenizer;

[0008] S2: adding 1% of the mass of the Phascolosoma esculenta homogenate flavor protease and 1% of the mass of the Phascolosoma esculenta homogenate neutral protease to the Phascolosoma esculenta homogenate, and hydrolyzing it at 55℃ for 6h to obtain an enzyme hydrolysate;

[0009] S3: The enzymatic hydrolysate is heated at 100 DEG C for 15 min for enzyme inactivation to obtain an enzyme-inactivated enzymatic hydrolysate;

[0010] S4: The enzyme-inactivated enzymatic hydrolysate is centrifuged at 10000 rpm / min for 10 min, and the supernatant is taken to obtain a crude sample solution of edible and delicious Phyllodoce lignosa protein peptide;

[0011] S5: The crude sample solution of edible and delicious Phyllodoce lignosa protein peptide is subjected to ultrafiltration separation under an ultrafiltration condition (operation pressure 0.4 MPa, operation temperature 4 DEG C), and ultrafiltration membranes with molecular weight cut-off of 10000 D and 5000 D are sequentially selected to obtain a Phyllodoce lignosa protein peptide solution as a permeate;

[0012] S6: The Phyllodoce lignosa protein peptide solution is pre-frozen at -80 DEG C for 10 h, and then continuously freeze-dried at -80 DEG C for 20 h to obtain the Phyllodoce lignosa protein peptide.

[0013] A Phyllodoce lignosa protein peptide prepared by the preparation method.

[0014] Application of the Phyllodoce lignosa protein peptide in preparation of a cell repair product.

[0015] Application of the Phyllodoce lignosa protein peptide in preparation of a whitening product.

[0016] The present application has the following advantages:

[0017] In the process of studying the Phyllodoce lignosa protein peptide, the present application first proves that the prepared Phyllodoce lignosa protein peptide has a whitening effect through a zebrafish model. The present application also determines that the prepared Phyllodoce lignosa protein peptide has a cell repair effect through experiments. Therefore, the present application proves through experiments that the freeze-dried product of the Phyllodoce lignosa protein peptide prepared by protease enzymolysis can promote cell repair and zebrafish whitening, and can be used for developing cosmetics, drugs, health products, food products and the like for promoting skin whitening, and the products can be a cosmetic series, a drug series. BRIEF DESCRIPTION OF DRAWINGS

[0018] Figure 1 Effects of different concentrations of Phyllodoce lignosa protein peptide on MRC-5 cell survival rate.

[0019] Figure 2 Effects of different concentrations of Phyllodoce lignosa protein peptide on MRC-5 cell scratch repair healing.

[0020] Figure 3 Test results of different concentrations of Phyllodoce lignosa protein peptide on zebrafish whitening.

[0021] Figure 4The results of the melanin inhibition rate test of different concentrations of Phascolosoma esculenta protein peptide on zebrafish. DETAILED DESCRIPTION

[0022] The technical solutions of the present application will be described clearly and completely below in combination with the embodiments of the present application. Obviously, the described embodiments are only some of the embodiments of the present application, but not all the embodiments. Based on the embodiments in the present application, all the other embodiments obtained by those skilled in the art without creative efforts belong to the scope of protection of the present application.

[0023] Embodiment 1

[0024] A preparation method of Phascolosoma esculenta protein peptide, comprising the following steps:

[0025] S1: Fresh and live Phascolosoma esculenta bodies are used as raw materials, washed clean with water, drained, cut into pieces, and added with water at a material-to-liquid mass ratio of 1:6, and homogenized into Phascolosoma esculenta homogenate by using a cell disrupter.

[0026] S2: Flavor protease (30000u / g) and neutral protease (50000u / g) are added to the Phascolosoma esculenta homogenate of step S1, the flavor protease is added in an amount of 1% of the mass of the homogenate, the neutral protease is added in an amount of 1% of the mass of the homogenate, and enzymolysis is performed at 55℃ for 6h to prepare an enzymolysis liquid.

[0027] S3: The enzymolysis liquid of step S2 is heated at 100℃ for 15min to fully inactivate the enzyme, and an inactivated enzyme enzymolysis liquid is prepared.

[0028] S4: The inactivated enzyme enzymolysis liquid of step S3 is centrifuged at 10000rpm / min for 10min to obtain a crude edible Phascolosoma esculenta protein peptide solution.

[0029] S5: The crude edible Phascolosoma esculenta protein peptide solution of step S4 is sequentially subjected to ultrafiltration separation by using ultrafiltration membranes with a molecular weight cut-off of 10000D and 5000D, and the ultrafiltration conditions are as follows: operating pressure 0.4MPa, operating temperature 4℃, and the permeate is the Phascolosoma esculenta protein peptide solution.

[0030] S6: The Phascolosoma esculenta protein peptide solution of step S5 is pre-frozen at-80℃ for 10h, and then continuously freeze-dried at-80℃ for 20h to prepare the Phascolosoma esculenta protein peptide.

[0031] The protein peptide powder of Phascolosoma esculenta obtained by the above method has a basic component protein content of 77%, a moisture content of 6.62%, and an amino acid content of 76.26 g / 100 g. Among them, glutamic acid is the most abundant amino acid in the protein peptide of Phascolosoma esculenta, accounting for 14.7% of the total amino acids, followed by arginine (8.15%), glycine (7.96%), and aspartic acid (7.24%). The total essential amino acids in the protein peptide of Phascolosoma esculenta account for 23.64% of the total amino acids. The molecular weight of the protein peptide of Phascolosoma esculenta is divided into four components, wherein the number average molecular weight of F1 is 5552 Da, accounting for 22.70%; the number average molecular weight of F2 is 1662 Da, accounting for 16.80%; the number average molecular weight of F3 is 702 Da, accounting for 22.20%; and the number average molecular weight of F4 is 185 Da, accounting for 38.3%.

[0032] Example 2

[0033] (1) The protein peptide of Phascolosoma esculenta was prepared according to the method provided in Example 1.

[0034] (2) Evaluation of the toxicity of different concentrations of the protein peptide of Phascolosoma esculenta on MRC-5 cells:

[0035] The CCK-8 method was used to determine the cytotoxicity of the protein peptide of Phascolosoma esculenta. 1×10 5 MRC-5 cells (human embryonic lung fibroblasts) were inoculated into a 96-well cell culture plate and placed in a cell incubator at 37°C with a CO2 concentration of 5% overnight. After the cells adhered, three groups, blank group, control group and sample group, were set up, with 6 replicates for each concentration in each group.

[0036] Blank group: no MRC-5 cells, add MEM medium;

[0037] Control group: containing MRC-5 cells, adding MEM medium;

[0038] Sample group: adding newly prepared MEM medium containing different concentrations of the protein peptide of Phascolosoma esculenta into the wells containing MRC-5 cells.

[0039] After adding the sample, the 96-well cell plate was placed in a cell incubator at 37°C with a CO2 concentration of 5%. After 24 h, 10 μL of CCK-8 was added to each well, and incubated at 37°C for 1 h. Finally, the absorbance value was measured at 450 nm to calculate the cell survival rate. The cell survival rate was calculated using the following formula:

[0040]

[0041] From Figure 1It was found that the safe concentration of *Sipunculus nudus* protein peptide in MRC-5 cells, determined through experiments, was 50-1000 μg / mL, under which the survival rate of MRC-5 cells exceeded 95%. The results indicate that *Sipunculus nudus* protein peptide showed no significant cytotoxicity to any cell type within the concentration range of 50-1000 μg / mL, making it a safe concentration for cell experiments.

[0042] Example 3

[0043] (1) Prepare the oral gnaphalium protein peptide according to the method provided in Example 1.

[0044] (2) Evaluation of the effect of different concentrations of palatable Sipunculus lablabi protein peptides on scratch repair in MRC-5 cells:

[0045] MRC-5 cells in logarithmic growth phase were injected with 5 × 10⁻⁶ cells. 4 Cells were seeded at a rate of 2 mL / well in 6-well plates and incubated at 37°C with 5% CO2 for 24 h to allow complete cell adhesion. Cell scars were then created in the culture plates using a cell scraper, the culture medium was removed, and the plates were washed twice with PBS buffer (0.01 M, pH 7.2). MEM medium containing different concentrations of *Sipunculus nudus* protein peptides (50, 100, 250, 500, 1000 μg / mL) was added, and photographs were taken. The plates were then incubated at 37°C with 5% CO2, and photographs were taken periodically (12 h and 24 h after drug addition). MEM medium without *Sipunculus nudus* protein peptides served as a control.

[0046] Depend on Figure 2 It was found that obvious scratch marks were still observed in the control group's MRC-5 cells after 24 hours. However, the scratch area in the MRC-5 cells treated with *Sipunculus nudus* protein peptides decreased after 24 hours. In particular, no obvious scratch marks remained at concentrations of 250 μg / mL and above. Therefore, the results indicate that *Sipunculus nudus* protein peptides promote the repair and proliferation of MRC-5 cells, and the intracellular repair and proliferation capacity of *Sipunculus nudus* protein peptides increases with increasing concentration. The cell repair and proliferation capabilities of this intervention offer promising applications, such as developing functional products like pharmaceuticals and cosmetics that promote skin wound healing and tissue repair. Therefore, based on the scratch test results, it can be further concluded that *Sipunculus nudus* protein peptides have the potential for application in pharmaceuticals, cosmetics, and other fields.

[0047] Example 4

[0048] (1) Prepare the oral gnaphalium protein peptide according to the method provided in Example 1.

[0049] (2) The maximum tolerated concentration (MTC) of different concentrations of Litopenaeus vannamei protein peptides on zebrafish was determined.

[0050] The previous night before collecting the embryos, healthy adult zebrafish were selected and paired in a 2:1 ratio of male to female in a mating tank, and the male and female zebrafish were separated by a partition. The next morning, after the light was turned on, the partition was removed, and the male and female fish were allowed to mate freely for 1-3 hours. The eggs produced by the mating of zebrafish were collected using a mesh spoon and placed in a 90 mm petri dish. 20 mL of prepared Litopenaeus vannamei protein peptides (0, 50 μg / mL, 100 μg / mL, 250 μg / mL, 500 μg / mL) were added to the 90 mm petri dish, and three parallel groups were set up. The petri dishes were incubated in a 28±0.5℃ constant temperature incubator.

[0051] The highest concentration group in which no 3dpf zebrafish larvae showed any death (no heartbeat) and other toxic effects (pericardial edema, trunk bending, no response to mechanical stimulation, unclear muscle texture, etc.) was determined as the MTC (Maximum tolerated concentration).

[0052] When the concentration of Litopenaeus vannamei protein peptides was 50 μg / mL, 100 μg / mL, and 250 μg / mL, no 3dpf zebrafish larvae showed any death (no heartbeat) and other toxic effects (pericardial edema, trunk bending, no response to mechanical stimulation, unclear muscle texture, etc.). When the concentration of Litopenaeus vannamei protein peptides was 500 μg / mL, 3dpf zebrafish larvae showed death. Therefore, 250 μg / mL of Litopenaeus vannamei protein peptides was the highest concentration group.

[0053] Example 5

[0054] (1) The Litopenaeus vannamei protein peptides were prepared according to the method provided in Example 1.

[0055] (2) Evaluation of whitening efficacy:

[0056] Zebrafish embryo preparation: the previous night, the embryos were collected, healthy adult zebrafish were selected, and the male and female zebrafish were paired at a ratio of 2:1 and placed in a mating tank, and the male and female zebrafish were separated by a partition. After the light was turned on the next morning, the partition was removed, and the male and female fish were allowed to mate freely for 1-3 hours. The fish in each tank were then checked for egg production, and the zebrafish embryos produced by mating were collected using a mesh spoon and placed in a 90 mm culture dish. The dish was then placed in a constant temperature incubator. Under a stereomicroscope, 6-8 hpf normally developed embryos were randomly selected and placed in a 6-well plate. After the water was absorbed with a plastic pipette, 3.0 mL of prepared Dugesia japonica protein peptide (0, 50 μg / mL, 100 μg / mL, 250 μg / mL) was added to each well of the 6-well cell culture plate. Three parallel groups were set up, and the plates were then covered with a cover plate and placed in a constant temperature incubator at 28±0.5°C. The distribution of melanin granules in the embryos at 48 hpf was observed and recorded under a stereomicroscope, and the zebrafish gray scale was analyzed using Image J software.

[0057] The results of the whitening effect of Dugesia japonica protein peptide on zebrafish are shown in Figure 3 As can be seen from Figure 3 , the normal group of zebrafish (without Dugesia japonica protein peptide intervention) had uniform distribution of surface melanin, and the eye melanin was obvious. Dugesia japonica protein peptide treatment can effectively reduce the surface melanin of zebrafish. When the concentration of Dugesia japonica protein peptide is 50-250 μg / mL, the surface melanin of zebrafish decreases with increasing concentration. When the concentration of Dugesia japonica protein peptide is 250 μg / mL, the surface melanin of zebrafish is the least. Figure 4 As can be seen from Figure 4 , the inhibition rate of Dugesia japonica protein peptide on the surface melanin of zebrafish is 52.19% when the concentration is 250 μg / mL, which is significantly higher than that of the 50 μg / mL (36.12%) and 100 μg / mL (29.39%) treatment groups. When the concentration of Dugesia japonica protein peptide is 50-250 μg / mL, the inhibition rate of Dugesia japonica protein peptide on melanin synthesis in vivo increases with increasing concentration. The inhibition rate of Dugesia japonica protein peptide on zebrafish melanin is consistent with the results of zebrafish appearance observation.

[0058] The above zebrafish experiment shows that Dugesia japonica protein peptide has a whitening effect and can be used to develop skin whitening cosmetics, drugs, etc. The products can be a series of cosmetics, drugs, etc. The products can be used in the form of application and oral administration, and long-term or long-term continuous use is recommended to achieve significant results.

Claims

1. A method for preparing a Phascolosoma esculenta protein peptide, characterized by comprising the steps of: The method comprises the following steps: ​ S1: using fresh and fresh Aplysia juliana as raw material, cleaning with water, draining water, cutting, adding 6 times of water of the raw material, and homogenizing into Aplysia juliana homogenate by a cell wall breaking machine; S2: adding 1% flavor protease and 1% neutral protease of the homogenate to the Aplysia juliana homogenate, and enzymolysis at 55℃ for 6h to obtain an enzymolysis liquid; S3: heating the enzymolysis liquid at 100℃ for 15min for enzyme inactivation treatment to obtain an enzyme inactivation enzymolysis liquid; S4: centrifuging the enzyme inactivation enzymolysis liquid at 10000rpm / min for 10min, taking the supernatant, and obtaining an edible Aplysia juliana protein peptide crude sample solution; S5: under the ultrafiltration condition, the Aplysia juliana protein peptide crude sample solution is sequentially separated by ultrafiltration membranes with a molecular weight cut-off of 10000D and 5000D, and the permeate is an Aplysia juliana protein peptide solution; S6: freeze-drying the Aplysia juliana protein peptide solution to obtain the Aplysia juliana protein peptide.

2. The method for preparing a palatable Sipunculus nudus protein peptide according to claim 1, characterized in that: The ultrafiltration condition is that the operation pressure is 0.4MPa and the operation temperature is 4℃.

3. The method according to claim 1, wherein the protein peptide is prepared from Phascolosoma esculenta.

3. The method according to claim 1, wherein the protein peptide is prepared from Phascolosoma esculenta. The freeze-drying condition is that pre-freezing at-80℃ for 10h, and then continuously freeze-drying at-80℃ for 20h.

4. An Aplysia juliana protein peptide prepared by the preparation method of claim 1.

5. Use of a Phascolosoma esculenta protein peptide according to claim 4 for the preparation of a cell repair product, characterized in that: The product is a cosmetic or a medicine.

6. Use of a Phascolosoma esculenta protein peptide according to claim 4 for the preparation of a whitening product, characterized in that: The product is a cosmetic or a medicine.

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