Algal protein peptide with anti-radiation activity and preparation method and application thereof

Abstract

The application discloses an algal protein peptide with anti-radiation activity and a preparation method and application thereof. The algal protein peptide is prepared by the following steps: adding pectinase and cellulase into red alga raw materials to release the algal protein by sol and lysis; adopting enzyme conversion to cut subunits to identify disulfide bonds and peptide bonds, so that a specific functional fragment component of the algal protein peptide is obtained; and through activity verification, the algal protein peptide with anti-radiation function is prepared in a targeted manner. The prepared algal protein peptide has a very strong protective effect on ultraviolet radiation HaCat cells, can effectively relieve cell DNA damage caused by UVB, improve cell survival rate under UVB irradiation, and reduce mortality. Therefore, the algal protein peptide can be applied to the preparation of food, cosmetics, drinks and other products, and has a good anti-radiation effect.

Description

Technical Field

[0001] This invention belongs to the field of biotechnology, specifically relating to an algal protein peptide with anti-radiation activity, its preparation method, and its application. Background Technology

[0002] Red algae (Rhodophyta) are collectively known as red algae, with thallus colors ranging from bright red, rose red, purplish red, to dark red. The vast majority of red algae grow in the ocean, are widely distributed, and have numerous species, estimated at approximately 558 genera and over 3700 species. They are further divided into two subclasses: Bangioideae and Florideae. Red algae have significant economic value, serving not only as food but also as raw materials in the medical, textile, and food industries. Red algae contain phycoerythrin (R-PE), hence their red color. Algal proteins (R-PE and B-PE) typically exist as 33 kDa oligomeric proteins composed of α, β, and γ subunits, with a molar ratio of α:β:γ generally of 6:6:1. Related data shows that the α and β subunits can form stable αβ monomers. Three αβ monomers polymerize 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. Currently, four phycobiliproteins have been identified: phycoerythrobilin (PEB), phycocyanobilin (PCB), phycourobilin (PUB), and phycoviolobilin (PVB). The chromophores of B-PE are similar to those of R-PE. Unlike R-PE and B-PE, the two trimers (αβ)3 are linked by a linking polypeptide L. R And without the γ subunit, and L RIt does not contain chromophores. C-PE-I is similar to the αβ monomers of B-PE. Studies have found that the amino acid sequence analysis of the α and β subunits of various PEs shows that although the number and types of amino acids differ, their three-dimensional structures are very similar. Regarding the immunomodulatory molecular effects of R-PE, he verified at the gene and protein expression levels that R-PE can activate the TLR4 / NF-κB signaling pathway and promote the proliferation and differentiation of CD4+ T cells. Specifically, R-PE upregulates the CD4+ / CD8+ ratio, increases the secretion of Th1, Th2, and Th17 cytokines and the expression levels of nuclear transcription factors T-bet, GATA3, and RORγt mRNA, inhibits the secretion of Treg cytokines and the expression level of nuclear transcription factor Foxp3 mRNA, and simultaneously increases the T-bet / GATA3 and RORγt / Foxp3 ratios, reversing the Th1 / Th2 and Th17 / Treg imbalance in immunosuppressed mice. These findings suggest that R-PE may maintain immune homeostasis and mitigate the immunosuppressive effects of HC through immune cell differentiation mediated by the TLR4 / NF-κB signaling pathway. However, currently, there are no methods for preparing radiation-resistant red algal protein peptides or their applications. Summary of the Invention

[0003] The purpose of this invention is to provide an algal protein peptide with anti-radiation activity, its preparation method, and its applications. This invention uses red algae containing algal proteins as raw material and directionally prepares an algal protein active peptide with anti-radiation function through online controlled biochemical engineering technology. This active peptide can be applied to the development of related functional products such as cosmetics and food and beverages with anti-radiation functions.

[0004] To achieve the above-mentioned objectives, the technical solution adopted by the present invention is as follows:

[0005] This invention provides a method for preparing algal protein peptides with anti-radiation activity, comprising the following steps:

[0006] (1) After rinsing the red algae raw material to remove impurities, grind it;

[0007] (2) After the raw material ground in step (1) is heated, agarase and cellulase are added to react, and the supernatant is taken by centrifugation;

[0008] (3) After concentrating the supernatant from step (2), salt out the solution, centrifuge to collect the precipitate, and then reconstitute it.

[0009] (4) Adjust the pH of the reconstituted solution in step (3), add papain to react, then adjust the pH again and repeat the addition of papain to react, centrifuge and take the supernatant.

[0010] (5) Concentrate the supernatant from step (4) to obtain a concentrated solution, dry it, collect the dry powder, and obtain algal protein peptides.

[0011] Furthermore, in step (1), the grinding is carried out using a colloid mill, and the mixture is transferred to the colloid mill with water at a solid-liquid ratio of 1:20-30 for 3-5 cycles.

[0012] Furthermore, in step (2), the amount of agarase used is 30-50 U / g and the amount of cellulase used is 20-30 U / g, based on the dry weight of the red algae raw material.

[0013] Furthermore, in step (3), 30% saturated ammonium sulfate is used for salting out.

[0014] Furthermore, in step (4), the pH of the reconstituted solution is first adjusted to 5.0, kept warm to 38°C, 200 U / ml papain is added, kept warm for 1 hour, the pH of the solution is then adjusted to 8.0, 100 U / ml papain is added, kept warm for 1 hour, heated to 80°C, cooled to 50°C, and the supernatant is collected by centrifugation.

[0015] Furthermore, in step (5), the concentration is carried out at 35°C using a double-effect concentrator until the soluble solids content is 20%-25%.

[0016] Furthermore, in step (6), the drying is carried out by spray drying, and the spray parameters are controlled as follows: air inlet temperature 150-160℃, air outlet temperature 90-99℃; the moisture content of the dry powder is <4%.

[0017] The present invention also provides an algal protein peptide prepared by the above preparation method, the amino acid sequence of which is: LKKFIADGNKR.

[0018] This invention also provides the application of the aforementioned algal protein peptide in the preparation of anti-radiation foods, beverages, and cosmetics.

[0019] Furthermore, the amount of the algal protein peptide used in cosmetics is 0.02%-0.2%.

[0020] This invention also provides an anti-radiation face cream, which is prepared using the aforementioned algal protein peptides; its formula, by weight ratio, is as follows:

[0021] 2-3 portions of algal protein peptides;

[0022] 5-10 parts glycerin;

[0023] Hyaluronic acid 1-5 parts;

[0024] 750-850 portions of purified water;

[0025] 80-100 parts of white oil;

[0026] 45-50 parts fenugreek seed oil;

[0027] 20-30 parts of surfactant

[0028] 5-10 parts of emulsified beeswax.

[0029] Compared with the prior art, the present invention has the following advantages and beneficial effects:

[0030] Phycobiliproteins (R-PE and B-PE) are typically oligomeric proteins composed of α, β, and γ subunits. By testing and selecting proteases that can both separate disulfide bonds and open subunits, and exhibit protein disulfide bond selectivity, and using the cell-killing rate of UVB radiation as an indicator, active protein peptides with UVB resistance are obtained. Phycobiliproteins (BPs) are hydrophilic pigment proteins with fluorescent properties, widely found in red and cyanobacteria. PBPs are composed of phycobiliin and apolipoproteins linked by covalent bonds, interconnected to form phycobilisomes, located on the outer surface of the photosynthetic membrane. There are three main types of phycobiliproteins: phycoerythrin (PE), phycocyanin (PC), and allophycocyanin (APC). PE is at the apex of the rod-shaped phycobilisome, PC is in the middle, and APC forms the core, attached to the phycobilisome. The reaction and energy transfer benefits sequentially from PE → PC → APC → chlorophyll, endowing the phycobiliprotein peptides with radiation resistance. This invention involves adding pectinase and cellulase to red algae raw materials to release red algal proteins through sol-gel and cytolysis processes. Then, an enzymatic method is used to cleave subunits and identify disulfide and peptide bonds to obtain red algal protein peptides with specific functional fragments. Furthermore, the activity of these peptides is verified to prepare radiation-resistant algal protein peptides. The obtained algal protein peptides are then tested for radiation resistance using human epidermal cells, demonstrating a strong protective effect against ultraviolet-irradiated HaCat cells. They effectively alleviate UVB-induced DNA damage, improve UVB-irradiated cell survival rates, and reduce mortality. Therefore, these algal protein peptides can be used to prepare radiation-resistant products such as food, cosmetics, and beverages. Attached Figure Description

[0031] Figure 1 The survival rate of cells protected against UVB was determined by different concentrations of the sample.

[0032] Figure 2 The effect of different concentrations of samples on CPD content in Hacat cells. Detailed Implementation

[0033] The technical solution of the present invention will be further described in detail with reference to the following specific examples.

[0034] Unless otherwise specified, the experimental methods used in the following examples are all conventional methods, and the materials and reagents used can be purchased from biological or chemical reagent companies.

[0035] Example 1

[0036] A method for preparing an algal protein peptide with anti-radiation activity includes the following steps:

[0037] (1) Red algae raw material processing: Rinse the red algae raw material to remove impurities, and transfer it to a colloid mill with water at a solid-liquid ratio of 1:20 for circulation 3 times for later use;

[0038] (2) Lysis treatment: Algal protein is released by sol and lysing by adding agarase and cellulase. Specifically, the raw material in step (1) is heated to 50°C, agarase is added at 30 U / g dry weight and cellulase is added at 20 U / g dry weight, and the mixture is stirred for 2 hours. The temperature is then raised to 80°C for 10 minutes, cooled, and centrifuged to obtain the supernatant lysate for later use.

[0039] (3) Algal protein extraction: The supernatant from step (2) is concentrated under reduced pressure to 1 / 3 volume, salted out with 30% saturated ammonium sulfate, centrifuged to remove the supernatant, the precipitate is reconstituted with 5 times the volume of distilled water, centrifuged to remove impurities, and the supernatant is set aside for use.

[0040] (4) Preparation of algal protein peptides: The specificity of active peptides is controlled by enzyme reaction conditions. The pH of the compound solution in step (3) is adjusted to 5.0 and kept at 38°C. Papain is added at 200 U / ml and kept at 1 h. The pH is adjusted to 8.0 and then papain is added at 100 U / ml and kept at 1 h. The solution is heated to 80°C, cooled to 50°C, and centrifuged to remove impurities, thus obtaining an extract containing anti-radiation active peptides.

[0041] (5) Concentration of algal protein peptide solution: This process uses a double-effect concentrator for low-temperature concentration, specifically maintaining the temperature at 35°C, until the soluble solids content is 20%;

[0042] (6) Preparation of anti-radiation active algal protein peptide extract: The concentrate from step (5) is spray-dried, and the spray parameters are controlled as follows: air inlet 150-160℃, air outlet 90-99℃; collect the dry powder, with a moisture content of <4%;

[0043] (7) Pack the dry powder from step (6) in an aluminum foil bag and store it in a dry and cool place to obtain anti-radiation active algal protein peptide.

[0044] Example 2

[0045] I. Quality Control and Testing of Anti-radiation Active Algal Protein Peptides

[0046] The sequence of the anti-radiation active algal protein peptide prepared in Example 1 was identified. The peptides were analyzed by capillary high-performance liquid chromatography (HPLC) and Orbitrap Elite™ mass spectrometry. After molecular weight determination, the peptide fragment with the highest abundance was selected and its sequence information was obtained by tandem mass spectrometry (MS / MS). Xcalibur software (Thermo Scientific MS software) and Max-Quant (1.6.2.10) were used to assist in the analysis of the raw mass spectrometry data. Byonic was used to search and analyze the target protein database, and the matching result was: a peptide of the β subunit (LKKFIADGNKR) of R-phycoerythrin; the specific steps and conditions are as follows:

[0047] 1. Experimental Materials

[0048] 1) Reagents and consumables:

[0049]

[0050] 2) Main instruments:

[0051]

[0052] 2. Sample pretreatment

[0053] 1) Dissolve the algal protein peptide dry powder sample prepared in Example 1 in ddH2O, add DTT solution to an appropriate amount of sample to make the final concentration 10 mmol / L, and reduce in a water bath at 56℃ for 1 h.

[0054] 2) Add IAA solution to make a final concentration of 50 mmol / L, and react in the dark for 40 min.

[0055] 3) Desalinate using a self-filled desalting column and evaporate the solvent in a vacuum centrifuge at 45°C.

[0056] 3. LC-MS / MS detection

[0057] A. Capillary liquid chromatography conditions:

[0058] 1) Pre-column: 300 μm id × 5 mm, packed with Acclaim PepMap RPLC C18, 5 μm, 100Å;

[0059] 2) Analytical column: 150 μm id × 150 mm, packed with Acclaim PepMap RPLC C18, 1.9 μm, 100 Å;

[0060] 3) Mobile phase A: 0.1% formic acid;

[0061] 4) Mobile phase B: 0.1% formic acid, 80% ACN;

[0062] 5) Flow rate: 600 nL / min;

[0063] 6) Analysis time for each component: 66 min;

[0064]

[0065] B. Mass Spectrometry Conditions

[0066] 1) Primary mass spectrometry parameters:

[0067] Resolution: 70,000;

[0068] AGCtarget: 3e6;

[0069] MaximumIT: 100ms;

[0070] Scan range: 300 to 1800 m / z;

[0071] 2) Secondary mass spectrometry parameters:

[0072] Resolution: 17,500;

[0073] AGCtarget: 1e5;

[0074] Maximum IT: 50ms;

[0075] TopN: 20;

[0076] NCE / steppedNCE: 28.

[0077] 4. Database retrieval

[0078] The raw mass spectrometry files were searched using Byonic to retrieve the target protein database, with the following search parameters:

[0079] 1) Fixed modifications: Carbamidomethyl (C);

[0080] 2) Variable modifications: Oxidation (M);

[0081] 3) Enzyme: Non specific;

[0082] 4) Maximum Missed Cleavages: 3;

[0083] 5) Peptide Mass Tolerance (PMT): 20 ppm;

[0084] 6) Secondary mass spectrometry error (Fragment Mass Tolerance): 0.02 Da.

[0085] II. Detection and Verification of the Anti-radiation Activity of Algal Protein Peptides

[0086] 1. Evaluation of the activity of algal protein peptides in protecting HaCat cells against UVB radiation.

[0087] 1.1 Experimental Materials

[0088] HaCat cells, DMEM high-glucose medium, fetal bovine serum, antibiotics, trypsin, glutamine, PBS, 96-well cell culture plates, cell culture dishes, sterile aluminum foil, and sterile blades, etc.

[0089] 1.2 Experimental Methods

[0090] UVB irradiation method: The irradiation was conducted in a dark-box ultraviolet analyzer at a wavelength of 302 nm and an irradiation intensity of 40 mJ / cm². 2 ;

[0091] Cell viability detection method: MTT assay.

[0092] 1.3 Experimental Samples and Concentrations

[0093] Experimental sample: Algal protein peptides prepared in Example 1;

[0094] Sample concentrations: 0.125 mg / mL, 0.25 mg / mL, 0.5 mg / mL, 1 mg / mL, 2 mg / mL.

[0095] 1.4 Experimental Grouping

[0096] Control group: HaCat cells were cultured normally and were not irradiated by UVB;

[0097] Model group: HaCat cells were cultured for 12 hours and then incubated in DMEM high-glucose medium for 24 hours before being irradiated with UVB at an intensity of 40 mJ / cm². 2 Then continue culturing for another 24 hours;

[0098] Sample group: HaCat cells were cultured for 12 hours and adhered to the culture vessel. After incubation in the sample for 24 hours, they were irradiated with UVB at an intensity of 40 mJ / cm². 2 Then continue culturing for another 24 hours.

[0099] 1.5 Cell Viability Calculation Method

[0100] Cell viability (%) = (absorbance of sample group - absorbance of blank group) / (absorbance of control group - absorbance of blank group) * 100.

[0101] Experimental results and analysis: such as Figure 1 As shown in Table 1, the model group cells at 40 mJ / cm 2 After UVB irradiation of high intensity, the cell viability significantly decreased to 38.82%, indicating that UVB irradiation adversely affected the normal growth of HaCat cells. Algal protein peptides effectively protected cells against the effects of UVB irradiation. Specifically, when the concentration of algal protein peptides was 0.125 mg / mL, after co-incubation for 24 hours, the cell viability significantly increased to 51.97%. With further increases in sample concentration, the effect of cell resistance to UVB irradiation was further enhanced, reaching a peak at an algal protein peptide concentration of 1 mg / mL, with a cell viability of 58.51%. The relative viability ratio indicates that algal protein peptides have a significant effect on protecting HaCat cells against UVB irradiation.

[0102] Table 1. Analysis of the effects of different concentrations of samples on cellular UVB protection.

[0103]

[0104] 2. Evaluation of the protective effect of algal protein peptides against UVB-induced DNA damage.

[0105] In daily work and life, skin is frequently exposed to sunlight, especially in summer when the weather is hot, ultraviolet radiation is strong, and the skin is exposed to sunlight for a longer period of time compared to other seasons, making it prone to ultraviolet damage. One of the hallmark damages of excessive ultraviolet exposure is sunburn. Sunburn is characterized by epidermal cell necrosis, reduced antigen presentation, and acute inflammation. Cellular DNA, as a natural chromophore, can absorb photons from UVB, causing nucleotide rearrangement. The energy-absorbing DNA bases (pyrimidines) combine with adjacent pyrimidines to form cyclobutanepyrimidine dimers (CPD, accounting for approximately 75% of photoproducts), ultimately inducing cell necrosis and forming sunburned cells.

[0106] 1.1 Experimental Materials

[0107] Human CPD ELISA kit, HaCat cells, DMEM high glucose medium, fetal bovine serum, penicillin antibodies, trypsin, glutamine, PBS, 96-well cell culture plate, cell culture dish, sterile aluminum foil, and sterile blade, etc.

[0108] 1.2 Experimental Methods

[0109] UVB irradiation method: The irradiation was conducted in a dark-box ultraviolet analyzer at a wavelength of 302 nm and an irradiation intensity of 40 mJ / cm². 2 ;

[0110] CPD content detection method: ELISA.

[0111] 1.3 Experimental Samples and Concentrations

[0112] Experimental sample: Algal protein peptides prepared in Example 1;

[0113] Sample concentrations: 0.25 mg / mL, 0.5 mg / mL, 1 mg / mL.

[0114] 1.4 Experimental Grouping

[0115] Control group: HaCat cells were cultured normally and were not irradiated by UVB;

[0116] Model group: HaCat cells were cultured for 12 hours and then adhered to the culture vessel. After incubation in DMEM complete medium for 24 hours, they were irradiated with UVB at an intensity of 40 mJ / cm². 2 Then continue culturing for another 24 hours;

[0117] Positive group: HaCat cells were cultured for 12 hours and then incubated with VC for 24 hours before being irradiated with UVB at an intensity of 40 mJ / cm². 2 Then continue culturing for another 24 hours;

[0118] Sample group: HaCat cells were cultured for 12 hours and adhered to the culture vessel. After incubation in the sample for 24 hours, they were irradiated with UVB at an intensity of 40 mJ / cm². 2 Then continue culturing for another 24 hours.

[0119] 1.5 Experimental Results and Analysis

[0120] like Figure 2 As shown, the model group cells were at 40 mJ / cm 2 After UVB irradiation of varying intensity, the CPD content in the model group CM was significantly increased compared to the blank control group CK, indicating successful modeling. Compared to the model group CM, the CPD content in cells from different concentration groups (0.25, 0.5, 1 mg / ml) was significantly decreased. P<0.001 The results showed a significant dose-dependent effect, indicating that algal peptides can effectively alleviate DNA damage in Hacat cells caused by UVB.

[0121] Example 3: Application of anti-radiation active algal protein peptides in cosmetics

[0122] The anti-radiation face cream is formulated with anti-radiation algae protein as the active ingredient. The usage range is 0.02-0.2% depending on the formula. The specific formula is as follows:

[0123] Aqueous phase composition:

[0124] The prepared algal protein peptide was 0.3 g / kg;

[0125] Glycerin 0.9 g / kg;

[0126] Hyaluronic acid 0.2 g / kg;

[0127] Purified water 80.6 g / kg;

[0128] Oil phase composition:

[0129] White oil 100 g / kg;

[0130] Fenugreek seed oil 45 g / kg;

[0131] Surfactant (sodium dodecylbenzenesulfonate) 25 g / kg;

[0132] Emulsified beeswax 10g / kg.

[0133] The preparation steps are as follows: Prepare the aqueous phase and oil phase according to the above proportions, taking 100 kg of face cream as an example:

[0134] Prepare the aqueous phase: Weigh 0.3 kg of the algal protein peptides prepared by the above process; 0.9 kg of glycerin; 0.2 kg of hyaluronic acid; and 80.6 kg of purified water, and 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, and mix well for later use. Dissolve the above oil and aqueous phase components at 86℃, and mix the prepared lipophilic and hydrophilic mixture evenly through a homogenizer. After cooling and settling, the anti-radiation face cream is obtained.

[0135] The above embodiments are only used to illustrate the technical solutions of the present invention, and are not intended to limit it. Although the present invention has been described in detail with reference to the foregoing embodiments, those skilled in the art can still modify the technical solutions described in the active algae protein peptide examples, or make equivalent substitutions for some of the technical features. Such modifications or substitutions do not cause the essence of the corresponding technical solutions to deviate from the spirit and scope of the technical solutions claimed by the present invention.

Claims

1. An algal protein peptide with anti-radiation activity, characterized in that, The amino acid sequence of the algal protein peptide is: LKKFIADGNKR; the preparation method of the algal protein peptide includes the following steps: (1) After rinsing the red algae raw material to remove impurities, grind it; (2) After the raw material ground in step (1) is heated, agarase and cellulase are added to react, and the supernatant is taken by centrifugation; 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 based on the dry weight of the red algae raw material. (3) After concentrating the supernatant from step (2), salting out the precipitate by centrifugation and then re-dissolving it; the salting out is carried out using 30% saturated ammonium sulfate. (4) Adjust the pH of the reconstituted solution in step (3) to 5.0, keep it warm to 38°C, add 200 U / ml papain, keep it warm for 1 h, then adjust the pH of the solution to 8.0, add 100 U / ml papain, keep it warm for 1 h, heat it to 80°C, cool it down to 50°C, centrifuge and take the supernatant. (5) Concentrate the supernatant from step (4) to obtain a concentrated solution, dry it, collect the dry powder, and obtain algal protein peptides.

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

3. The algal protein peptide according to claim 1, characterized in that, In step (5), the concentration is carried out at 35°C using a double-effect concentrator until the soluble solids content is 20%-25%.

4. The application of the algal protein peptide according to claim 1 in the preparation of anti-radiation foods, beverages, and cosmetics, characterized in that, The amount of the algal protein peptide used in cosmetics is 0.02%-0.2%.

5. An anti-radiation face cream, characterized in that, The anti-radiation face cream is prepared using the algal protein peptide described in claim 1; its formula, by weight, is as follows: 2-3 portions of algal protein peptides; 5-10 parts glycerin; Hyaluronic acid 1-5 parts; 750-850 servings of purified water; 80-100 parts of white oil; 45-50 parts fenugreek seed oil; 20-30 parts of surfactant 5-10 parts of emulsified beeswax.

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