Algae protein peptide with anti-radiation activity as well as preparation method and application thereof

Abstract

The invention discloses an algae protein peptide with anti-radiation activity and a preparation method and application thereof. The method comprises the following steps: adding pectinase and cellulase into a red algae raw material, and carrying out sol dissolving and cell dissolving to release red algae protein; the preparation method comprises the following steps: by taking a red algae protein peptide as a raw material, cutting a subunit by virtue of enzymatic conversion to identify a disulfide bond and a peptide bond, so as to obtain the red algae protein peptide with a specific functional fragment component, and directionally preparing the algae protein peptide with an anti-radiation function through activity verification, so that the prepared algae protein peptide has an extremely strong protection effect on ultraviolet irradiation HaCat cells, can effectively relieve cell DNA damage caused by UVB, and can be used for preparing the anti-radiation algae protein peptide. The cell survival rate of UVB irradiation is improved, and the death rate is reduced, so that the algae protein peptide can be applied to preparation of products such as food, cosmetics and drinks, and has a good anti-radiation effect.

Description

Technical Field

[0001] The invention belongs to the field of biotechnology, and specifically relates to an algae protein peptide with anti-radiation activity and a preparation method and application thereof. Background Art

[0002] Rhodophyta algae are collectively called red algae, and the algae are bright red, rose red, purple red and dark red. Most of the red algae grow in the ocean, with a wide distribution and many species. According to statistics, there are about 558 genera and more than 3,700 species, which are divided into two subclasses: Bangioideae and Florideae. Red algae have important economic value. In addition to being edible, they are also raw materials for industries such as medicine, textiles, and food. Red algae contain phycoerythrin (R-PE), so they are often red. Phycoerythrin (R-PE and B-PE) usually exists in the form of oligomeric proteins of 33 kDa composed of α, β and γ subunits, and the molar ratio between the three subunits is generally α: β: γ = 6: 6: 1. Related data show that the α subunit and the β subunit can form a stable αβ monomer. Three αβ monomers are connected end to end to form a disc-shaped trimer (αβ)3. The γ subunit connects two trimers to form a hexamer (αβ)6γ or (αβ)3γ(αβ)3. At present, four types of phycobilins have been found in phycobiliproteins, namely phycoerythrobilin (PEB), phycocyanobilin (PCB), phycourobilin (PUB) and phycobilipurpurin (PVB). The chromophore type of B-PE is similar to that of R-PE. Unlike R-PE and B-PE, the two trimers (αβ)3 are connected by a connecting polypeptide L R , but not the γ subunit, and L RIt does not contain chromophores. C-PE-Ⅰ is similar to the αβ monomer of B-PE. The study found that the amino acid sequence analysis of the α and β subunits of various PEs showed that although there were differences in the number and type of amino acids, the three-dimensional structure was very similar. The immunomodulatory molecular role of R-PE He verified that R-PE can activate the TLR4 / NF-κB signaling pathway at the gene and protein expression levels, and promote the proliferation and differentiation of CD4+ T cells. Specifically, R-PE upregulates the ratio of CD4+ / CD8+, increases the secretion of Th1, Th2 and Th17 cytokines, and the expression level of mRNA of nuclear transcription factors T-bet, GATA3 and RORγt, inhibits the secretion of Treg cytokines and the expression level of mRNA of nuclear transcription factor Foxp3, and increases the ratio of T-bet / GATA3 and RORγt / Foxp3, reversing the imbalance of Th1 / Th2 and Th17 / Treg in immunosuppressive mice. These indicate that R-PE may maintain the body's immune homeostasis and reduce the immunosuppressive effect of HC on the body through the differentiation of immune cells mediated by the TLR4 / NF-κB signaling pathway. However, there is currently no preparation method for red algae protein peptides with anti-radiation function and related active applications. Summary of the invention

[0003] The purpose of the present invention is to provide an algae protein peptide with anti-radiation activity and a preparation method and application thereof. The present invention uses red algae containing algae protein as raw material, and uses online controlled biochemical engineering technology to directionally prepare algae protein active peptides with anti-radiation function. The active peptides can be used in the development of related functional products such as cosmetics, food and beverages with anti-radiation function.

[0004] In order to achieve the above-mentioned invention object, the technical solution adopted by the present invention is as follows: The present invention provides a method for preparing an algae protein peptide having anti-radiation activity, which comprises the following steps: (1) Rinse the red algae raw material to remove impurities and then grind it; (2) After the raw material ground in step (1) is heated, agarase and cellulase are added to react, and the supernatant is collected by centrifugation; (3) Concentrating the supernatant of step (2) and performing salting out, centrifuging to obtain the precipitate and then redissolving it; (4) adjusting the pH value of the reconstituted solution of step (3), adding papain for reaction, then adjusting the pH again and repeatedly adding papain for reaction, and centrifuging to obtain the supernatant; (5) Concentrating the supernatant of step (4), drying the concentrated solution, collecting the dry powder, and obtaining the algae protein peptide.

[0005] Furthermore, in the step (1), grinding is performed using a colloid mill, and the mixture is transferred into the colloid mill with water at a solid-to-liquid ratio of 1:20-30 and circulated for 3-5 times.

[0006] Furthermore, in the step (2), based on the dry weight of the red algae raw material, the amount of agarase used is 30-50 U / g, and the amount of cellulase used is 20-30 U / g.

[0007] Furthermore, in step (3), 30% saturation ammonium sulfate is used for salting out.

[0008] Furthermore, in step (4), the pH of the solution is first adjusted to 5.0, kept warm at 38°C, 200 U / ml papain is added, and kept warm for 1 hour, then the pH of the solution is adjusted to 8.0, 100 U / ml papain is added, and kept warm for 1 hour, heated to 80°C, cooled to 50°C, and centrifuged to obtain the supernatant.

[0009] Furthermore, in the step (5), the concentration is carried out at 35° C. using a double-effect concentrator to concentrate the soluble solids to 20%-25%.

[0010] Furthermore, in step (6), the drying is carried out by spray drying, and the spray parameters are controlled to be 150-160°C for inlet air and 90-99°C for outlet air; the moisture content of the dry powder is <4%.

[0011] The present invention also provides an algae protein peptide prepared by the preparation method, and its amino acid sequence is: LKKFIADGNKR.

[0012] The present invention also provides the use of the algae protein peptide in the preparation of radiation-resistant food, beverage and cosmetics.

[0013] Furthermore, the amount of the algae protein peptide used in cosmetics is 0.02%-0.2%.

[0014] The present invention also provides an anti-radiation facial cream, which is prepared by using the algae protein peptide; its formula is as follows by weight ratio: 2-3 parts of algae protein peptide; Glycerin 5-10 parts; 1-5 parts of hyaluronic acid; 750-850 parts of purified water; 80-100 parts of white oil; Fenugreek seed oil 45-50 parts; Surfactant 20-30 parts Emulsified beeswax 5-10 parts.

[0015] Compared with the prior art, the present invention has the following advantages and beneficial effects: Phycobiliproteins (R-PE and B-PE) are usually oligomeric proteins composed of α, β and γ subunits. Through testing, we selected proteases that can separate disulfide bond-opening subunits and have protein disulfide bond selectivity. Taking the cell killing rate of UVB radiation source as an indicator, we obtained active protein peptides with UVB resistance. Phycobiliproteins (PBPs) are hydrophilic pigment proteins with fluorescent properties, which are widely found in red algae and cyanobacteria. PBPs are composed of phycobiliproteins and apolipoproteins bound by covalent bonds, which are connected to each other to form phycobilisomes, located on the outer surface of photosynthetic membranes. There are three main types of phycobiliproteins: phycoerythrin (PE), phycocyanin (PC) and allophycocyanin (APC). PE is the top of the rod-shaped phycobilisome, PC is in the middle, and APC forms the core attached to the reaction and energy transfer proceeds from PE→PC→APC→chlorophyll, giving it the radiation resistance of phycobiliprotein peptides. The present invention adds pectinase and cellulase to the red algae raw material to perform sol and lysis to release red algae protein; then adopts enzymatic conversion to cut subunits to identify disulfide bonds and peptide bonds to obtain red algae protein peptides of specific functional fragment components; then, through activity verification, the phycoprotein peptides with radiation resistance function are prepared directionally; the prepared phycoprotein peptides are tested and verified for radiation resistance through human epidermal cells, and the obtained phycoprotein peptides have a strong protective effect on ultraviolet irradiated HaCat cells, can effectively alleviate cell DNA damage caused by UVB, improve the survival rate of UVB-irradiated cells, and reduce the mortality rate; therefore, the phycoprotein peptides can be used to prepare products with radiation resistance, such as food, cosmetics, and beverages. BRIEF DESCRIPTION OF THE DRAWINGS

[0016] Figure 1 The survival rate of cells against UVB of samples with different concentrations.

[0017] Figure 2 The effect of samples with different concentrations on the CPD content of Hacat cells. DETAILED DESCRIPTION

[0018] The technical solution of the present invention is further described in detail with reference to the following specific examples.

[0019] In the following examples, unless otherwise specified, the experimental methods used are all conventional methods, and the materials and reagents used can be purchased from biological or chemical reagent companies.

[0020] Example 1 A method for preparing an algae protein peptide having anti-radiation activity comprises the following steps: (1) Processing of red algae raw materials: rinse the red algae raw materials to remove impurities, and transfer them into the colloid mill with water at a solid-liquid ratio of 1:20 for 3 cycles for standby use; (2) Lysis treatment: Sol and lyse the algae protein by adding agarase and cellulase. Specifically, the raw material of step (1) is heated to 50°C, 30 U / g agarase and 20 U / g cellulase are added at dry weight, stirred and reacted for 2 hours, heated to 80°C for 10 minutes, cooled, and centrifuged to obtain the supernatant lysate for later use; (3) Extraction of algal protein: The supernatant of step (2) was concentrated under reduced pressure to 1 / 3 of its volume, and then salted out with 30% saturated ammonium sulfate. The supernatant was discarded by centrifugation, and the precipitate was re-dissolved with 5 times the volume of distilled water, and then centrifuged to remove impurities. The supernatant was set aside; (4) Preparation of algae protein peptides: The specificity of the active peptides was controlled by using enzyme reaction conditions. The pH of the reconstituted solution of step (3) was adjusted to 5.0 and kept warm at 38°C. Papain was added at 200 U / ml and kept warm for 1 hour. The pH was adjusted to 8.0. Papain was then added at 100 U / ml and kept warm for 1 hour. The solution was heated to 80°C, cooled to 50°C, and centrifuged to remove impurities to obtain an extract containing anti-radiation active peptides. (5) Concentration of algae protein peptide liquid: This process uses a double-effect concentrator for low-temperature concentration, specifically maintaining the temperature at 35°C and concentrating until the soluble solids are 20%; (6) Preparation of algae protein peptide extract containing anti-radiation activity: spray drying the concentrated solution of step (5), spray parameter control: inlet air 150-160°C, outlet air 90-99°C; collect dry powder, moisture <4%; (7) The dry powder of step (6) is packaged in an aluminum foil bag and stored in a dry and cool place to obtain an anti-radiation active algae protein peptide.

[0021] Example 2 1. Quality Control Testing of Anti-Radiation Active Algae Protein Peptides The sequence of the anti-radiation active algae protein peptide prepared in Example 1 was identified. The polypeptide was subjected to capillary high performance liquid chromatography (HPLC) spectrometer and Orbitrap Elite™ mass spectrometer. After molecular mass determination, the polypeptide fragment with the highest abundance was selected, and the sequence information was obtained by tandem mass spectrometry (MS / MS) analysis. Xcalibur software (Thermo Scientific MS software) and Max-Quant (1.6.2.10) were used to assist the mass spectrometry raw file, and Byonic was used to search and analyze the target protein database. The matching result was: the polypeptide of the β subunit of R-phycoerythrin (LKKFIADGNKR); the specific steps and conditions were as follows: 1. Experimental Materials 1) Reagents and consumables:

[0022] 2) Main instruments:

[0023] 2. Sample pretreatment 1) The dry powder sample of the algae protein peptide prepared in Example 1 was dissolved in ddH2O, and DTT solution was added to an appropriate amount of the sample to make the final concentration of 10 mmol / L, and reduced in a 56°C water bath for 1 h.

[0024] 2) Add IAA solution to a final concentration of 50 mmol / L and react for 40 min in the dark.

[0025] 3) Desalt using a self-packed desalting column and evaporate the solvent in a vacuum centrifugal concentrator at 45°C.

[0026] 3. LC-MS / MS detection A. Capillary liquid chromatography conditions: 1) Pre-column: 300 μm id × 5 mm, packed with Acclaim PepMap RPLC C18, 5 μm, 100Å; 2) Analytical column: 150 μm id × 150 mm, packed with Acclaim PepMap RPLC C18, 1.9 μm, 100Å; 3) Mobile phase A: 0.1% formic acid; 4) Mobile phase B: 0.1% formic acid, 80% ACN; 5) Flow rate: 600 nL / min; 6) Analysis time for each component: 66min;

[0027] B. Mass spectrometry conditions 1) Primary mass spectrometry parameters: Resolution: 70,000; AGCtarget: 3e6; MaximumIT: 100ms; Scanrange: 300 to 1800m / z; 2) Secondary mass spectrometry parameters: Resolution: 17,500; AGCtarget: 1e5; MaximumIT: 50ms; TopN: 20; NCE / steppedNCE:28.

[0028] 4. Database search The mass spectrometry raw file was searched against the target protein database using Byonic, and the search parameters were as follows: 1) Fixed modifications: Carbamidomethyl (C); 2) Variable modifications: Oxidation (M); 3) Enzyme: Non specific; 4) Maximum Missed Cleavages: 3; 5) Peptide Mass Tolerance: 20 ppm; 6) Fragment Mass Tolerance: 0.02Da.

[0029] 2. Detection and verification of the anti-radiation activity of algae protein peptides 1. Evaluation of the activity of algae protein peptide in protecting HaCat cells against UVB irradiation 1.1 Experimental Materials HaCat cells, DMEM high-glucose medium, fetal bovine serum, double antibody, trypsin, glutamine, PBS, 96-well cell culture plates, cell culture dishes, sterile tin foil and sterile blades, etc.

[0030] 1.2 Experimental methods UVB irradiation method: carried out in a dark box UV analyzer, with an irradiation wavelength of 302nm and an irradiation intensity of 40mJ / cm 2 ; Cell viability detection method: MTT method.

[0031] 1.3 Experimental samples and concentrations Experimental sample: algae protein peptide prepared in Example 1; Sample concentration: 0.125 mg / mL, 0.25 mg / mL, 0.5 mg / mL, 1 mg / mL, 2 mg / mL.

[0032] 1.4 Experimental Grouping Control group: HaCat cells were cultured normally without UVB irradiation; Model group: HaCat cells were cultured for 12 hours and adhered to the wall. They were then incubated in DMEM high-glucose medium for 24 hours and irradiated with UVB at an intensity of 40 mJ / cm 2 , and then continue to culture for 24h; Sample group: HaCat cells were cultured for 12 hours and adhered to the wall. After the samples were incubated for 24 hours, they were irradiated with UVB at an intensity of 40 mJ / cm 2 , and then continue to culture for 24h.

[0033] 1.5 Calculation method of cell viability Cell survival rate (%) = (absorbance value of sample group - absorbance value of blank group) / (absorbance value of control group - absorbance value of blank group) * 100.

[0034] Experimental results and analysis: Figure 1 As shown in Table 1, the cells in the model group were exposed to 40 mJ / cm 2 After high-intensity UVB irradiation, the cell survival rate dropped significantly to 38.82%, indicating that UVB irradiation has an adverse effect on the normal growth of HaCat cells. Algae protein peptide can effectively protect cells from the effects of UVB irradiation. Specifically, when the concentration of algae protein peptide is 0.125 mg / mL, after 24 hours of co-incubation, the cell survival rate increased significantly to 51.97%. As the sample concentration is further increased, the effect of cell resistance to UVB irradiation is further enhanced, reaching a peak at a concentration of 1 mg / mL, with a cell survival rate of 58.51%; the relative survival rate ratio shows that algae protein peptide has a significant effect on protecting HaCat cells from UVB irradiation.

[0035] Table 1 Analysis of the results of cell anti-UVB by samples of different concentrations

[0036] 2. Evaluation of the protective effect of algae protein peptides on DNA damage caused by UVB irradiation In daily work and life, the skin is often exposed to sunlight, especially in the summer when the weather is hot and the ultraviolet rays are strong. The skin is exposed to sunlight for a longer time than in other seasons, which makes it easy to form ultraviolet damage. One of the hallmark damages of excessive exposure of the skin to ultraviolet rays is the sunburn reaction. The characteristics of the sunburn reaction are epidermal cell necrosis, reduced antigen presentation and the occurrence of acute inflammation. As a natural chromophore, the DNA in the cell nucleus can absorb photons from UVB and rearrange the nucleotide structure. The pyrimidine of the DNA base that absorbs the energy can combine with the adjacent pyrimidine to form cyclobutanepyrimidine dimer (CPD, accounting for about 75% of the photoproducts), which eventually induces cell necrosis and forms sunburned cells.

[0037] 1.1 Experimental Materials Human CPD ELISA KIT, HaCat cells, DMEM high glucose medium, fetal bovine serum, double antibody, trypsin, glutamine, PBS, 96-well cell culture plates, cell culture dishes, sterile tin foil and sterile blades, etc.

[0038] 1.2 Experimental methods UVB irradiation method: carried out in a dark box UV analyzer, with an irradiation wavelength of 302nm and an irradiation intensity of 40mJ / cm 2 ; CPD content detection method: ELISA.

[0039] 1.3 Experimental samples and concentrations Experimental sample: algae protein peptide prepared in Example 1; Sample concentration: 0.25mg / mL, 0.5mg / mL, 1mg / mL.

[0040] 1.4 Experimental Grouping Control group: HaCat cells were cultured normally without UVB irradiation; Model group: HaCat cells were cultured for 12 hours and adhered to the wall. They were then incubated in DMEM complete medium for 24 hours and irradiated with UVB at an intensity of 40 mJ / cm 2 , and then continue to culture for 24h; Positive group: HaCat cells were cultured for 12 hours and adhered to the wall. They were then incubated with VC for 24 hours and irradiated with UVB at an intensity of 40 mJ / cm 2 , and then continue to culture for 24h; Sample group: HaCat cells were cultured for 12 hours and adhered to the wall. After the samples were incubated for 24 hours, they were irradiated with UVB at an intensity of 40 mJ / cm 2 , and then continue to culture for 24h.

[0041] 1.5 Experimental Results and Analysis like Figure 2 As shown, the cells in the model group were exposed to 40 mJ / cm 2 After irradiation with high-intensity UVB, the CPDs content of CM in the model group increased significantly compared with the blank control group CK, indicating that the modeling was successful. Compared with the CM in the model group, the CPDs content of cells in the different concentration sample groups (0.25, 0.5, 1 mg / ml) decreased significantly ( P<0.001 ), and showed significant dose dependence, indicating that algae protein peptide can effectively alleviate the DNA damage of Hacat cells caused by UVB.

[0042] Example 3: Application of Algae Protein Peptide with Anti-Radiation Activity in Cosmetics The preparation of the anti-radiation cream is a raw material formula with anti-radiation algae protein as the active ingredient. The usage range is 0.02-0.2% according to the formula. The specific formula is as follows: Water phase composition: Prepared algae protein peptide 0.3 g / kg; Glycerol 0.9 g / kg; Hyaluronic acid 0.2 g / kg; Pure water 80.6 g / kg; Oil phase composition: White oil 100 g / kg; Fenugreek seed oil 45 g / kg; Surfactant (sodium dodecylbenzene sulfonate) 25 g / kg; Emulsified beeswax 10g / kg.

[0043] The preparation steps are as follows: prepare the water phase and the oil phase according to the above ratios, taking 100 kg of cream as an example: Prepare the aqueous phase: weigh 0.3 kg of the algae protein peptide prepared by the above process; 0.9 kg of glycerol; 0.2 kg of hyaluronic acid; 80.6 kg of purified water, mix well for later use; prepare the oil phase: weigh 10 kg of white oil, 4.5 kg of fenugreek seed oil, 2.5 kg of sodium dodecylbenzene sulfonate, and 1 kg of emulsified beeswax, mix well for later use; dissolve the above oil phase and aqueous phase components at 86° C., mix the prepared lipophilic and hydrophilic mixture evenly with a homogenizer; and prepare the anti-radiation cream after cooling down.

[0044] The above embodiments are only used to illustrate the technical solutions of the present invention, rather than to limit the same. Although the present invention has been described in detail with reference to the above embodiments, it is still possible for a person skilled in the art to modify the technical solutions described in the active algae protein peptide examples, or to replace some of the technical features thereof by equivalent means. However, these modifications or replacements do not deviate the essence of the corresponding technical solutions from the spirit and scope of the technical solutions claimed to be protected by the present invention.

Claims

1. A method for preparing an algae protein peptide having anti-radiation activity, characterized in that: The following steps are involved: (1) Rinse the red algae raw material to remove impurities and then grind it; (2) After the raw material ground in step (1) is heated, agarase and cellulase are added to react, and the supernatant is collected by centrifugation; (3) Concentrating the supernatant of step (2) and performing salting out, centrifuging to obtain the precipitate and then redissolving it; (4) adjusting the pH value of the reconstituted solution of step (3), adding papain for reaction, then adjusting the pH again and repeatedly adding papain for reaction, and centrifuging to obtain the supernatant; (5) Concentrating the supernatant of step (4), drying the concentrated solution, collecting the dry powder, and obtaining the algae protein peptide.

2. The preparation method according to claim 1, characterized in that: In the step (1), grinding is carried out using a colloid mill, and the solid is transferred into the colloid mill with water at a solid-liquid ratio of 1:20-30 and circulated for 3-5 times.

3. The preparation method according to claim 1, characterized in that: In the step (2), based on the dry weight of the red algae raw material, the amount of agarase used is 30 U / g-50 U / g, and the amount of cellulase used is 20 U / g-30 U / g.

4. The preparation method according to claim 1, characterized in that: In the step (3), 30% saturation ammonium sulfate is used for salting out.

5. The preparation method according to claim 1, characterized in that: In the step (4), the pH of the solution is first adjusted to 5.0, kept warm at 38°C, 200 U / ml papain is added, and kept warm for 1 hour. The pH of the solution is then adjusted to 8.0, 100 U / ml papain is added, and kept warm for 1 hour. The solution is heated to 80°C, cooled to 50°C, and centrifuged to obtain the supernatant.

6. The preparation method according to claim 1, characterized in that: In the step (5), the concentration is carried out at 35° C. using a double-effect concentrator until the soluble solid content is 20%-25%.

7. The algae protein peptide prepared by the preparation method according to any one of claims 1 to 6, characterized in that: The amino acid sequence of the algae protein peptide is: LKKFIADGNKR.

8. Use of the algae protein peptide according to claim 7 in the preparation of radiation-resistant foods, beverages and cosmetics.

9. The use according to claim 8, characterized in that: The amount of the algae protein peptide used in cosmetics is 0.02%-0.2%.

10. An anti-radiation cream, characterized in that: The anti-radiation cream is prepared by using the algae protein peptide according to claim 7; its formula is as follows by weight: 2-3 parts of algae protein peptide; Glycerin 5-10 parts; 1-5 parts of hyaluronic acid; 750-850 parts of purified water; 80-100 parts of white oil; Fenugreek seed oil 45-50 parts; Surfactant 20-30 parts Emulsified beeswax 5-10 parts.

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