Preparation method and application of selenium-enriched spirulina active peptide

Abstract

The invention discloses a preparation method of selenium-enriched spirulina active peptide, and belongs to the field of biological medicine. The selenium-enriched spirulina is directly subjected to enzymolysis through a one-step enzymolysis method, parameters such as enzyme types, a material-liquid ratio, pH, temperature and time are optimized, the hydrolysis degree and the selenium content are remarkably improved, and the obtained selenium peptide is higher in antioxidant activity. The active peptide prepared by the invention contains 80 selenopeptide fragments, and can effectively inhibit inflammatory factors, repair intestinal barriers and reduce the DAI score, thereby improving the symptoms of ulcerative colitis.

Description

Technical Field

[0001] The present invention relates to the field of biomedicine, and specifically to a preparation method of selenium-enriched spirulina active peptides (SeSPs) and their application in the treatment of ulcerative colitis. Background Art

[0002] As a major subtype of inflammatory bowel disease, the global incidence of ulcerative colitis has been continuously rising in the past decade. This disease is characterized by continuous ulcers in the colonic mucosa layer, and the clinical manifestations are recurrent diarrhea, bloody stools, and abnormally elevated oxidative stress indicators. Currently, drugs for treating this disease have been developed clinically, such as sulfasalazine, corticosteroids, and immunosuppressants, etc., but there are still common problems such as significant drug side effects, insufficient efficacy persistence, and high treatment costs. Developing food-derived bioactive substances with both biosafety and targeted therapeutic effects has become an important research direction in this field.

[0003] Selenium is an essential trace element for the human body, mainly existing in selenoproteins in the form of selenocysteine, and plays various physiological functions such as antioxidant, anti-cancer, anti-inflammatory, and immunomodulatory effects. Existing research mainly focuses on the in vitro antioxidant properties of selenium peptides (such as DPPH free radical scavenging rate), and it is found that selenium peptides have both the biological activities of selenium and peptides, but there are few intervention studies on specific diseases for selenium peptides. Especially in the field of ulcerative colitis treatment, although some scholars have explored the protective effects of selenium-containing compounds or active peptides on colitis in recent years, there is still no reported systematic research on the efficacy of selenium peptides through regulating the intestinal oxidative stress pathway.

[0004] As a natural selenium-enriched biological carrier, the preparation and application of active peptides from spirulina have always been a research hotspot in the field of biotechnology. The traditional preparation of selenium-enriched active peptides mostly adopts the "two-step" process, that is, first, selenium-enriched proteins are separated through steps such as solvent extraction and salting-out precipitation, and then the purified proteins are enzymatically hydrolyzed. This process has obvious defects: First, in the protein separation stage, selenium is easily combined with sulfhydryl compounds to form insoluble complexes, resulting in a significant reduction in the selenium retention rate of the final product, and the active peptide components are prone to oxidative denaturation during the multi-step purification process; Second, the separate implementation of the separation and enzymatic hydrolysis processes leads to an extended production cycle, accompanied by a significant increase in energy consumption and production costs.

[0005] In view of the demand for highly efficient and safe preparations in the field of ulcerative colitis treatment, as well as the problems of activity loss and low process efficiency existing in the existing spirulina selenium peptide preparation technology, it is of great significance to develop spirulina selenium peptides that can simultaneously achieve high-efficiency retention of selenium elements, simplify the production process, and have a clear intestinal oxidative stress regulation function. Summary of the Invention

[0006] The object of the present invention is to provide a preparation method of selenium-enriched spirulina active peptides, which directly enzymatically hydrolyzes selenium-enriched spirulina by one-step enzymatic hydrolysis, optimizes parameters such as enzyme type, solid-liquid ratio, pH, temperature and time, and significantly improves the degree of hydrolysis and the selenium content of selenium peptides. The obtained active peptides contain 80 selenium-containing peptide segments (43 SeC and 37 SeM), which can effectively inhibit inflammatory factors, repair the intestinal barrier, and reduce the DAI score, thereby improving the symptoms of ulcerative colitis.

[0007] A preparation method of selenium-enriched spirulina active peptides (SeSPs), which adopts one-step enzymatic hydrolysis and comprises the following steps:

[0008] (a) Mix selenium-enriched spirulina with water at a solid-liquid mass ratio of 1:20 - 1:60;

[0009] (b) Add protease accounting for 2 - 6% of the mass of selenium-enriched spirulina, and carry out enzymatic hydrolysis at pH 7.5 - 10 and a temperature of 45 - 60 °C for 1 - 3 hours;

[0010] (c) Inactivate the enzyme and then centrifuge to obtain the supernatant;

[0011] (d) Filter the supernatant through an ultrafiltration membrane with a molecular weight cut-off of 3 - 10 kDa, and obtain selenium-enriched spirulina active peptides after freeze-drying.

[0012] Preferably, the protease is alkaline protease.

[0013] Preferably, the solid-liquid ratio is 1:40.

[0014] Preferably, the enzymatic hydrolysis conditions are: pH 8 - 9, temperature 50 - 55 °C, and enzymatic hydrolysis time 2 - 2.5 hours.

[0015] Preferably, the molecular weight cut-off of the ultrafiltration membrane is 5 kDa.

[0016] Preferably, the enzyme addition amount is 4% of the mass of selenium-enriched spirulina.

[0017] It is detected that the prepared selenium-enriched spirulina active peptides contain 80 selenium-containing amino acids, including 43 selenocysteine (SeC) and 37 selenomethionine (SeM).

[0018] Furthermore, a mouse model of ulcerative colitis was constructed using 2.5% dextran sulfate sodium salt, and it was found that selenium-enriched spirulina active peptides can significantly reduce the DAI score and improve the organ index, reduce the cytokine level in the colon, and at the same time repair the intestinal barrier function to maintain the integrity of the intestinal mucosa, thereby playing a therapeutic role in ulcerative colitis.

[0019] The beneficial effects of the present invention are:

[0020] The present invention uses a one-step enzymatic hydrolysis method to prepare selenium-enriched spirulina active peptides. This method has the advantages of high selenium recovery rate, short production cycle, and low cost. There are many types and high contents of selenium peptides in the product, which is beneficial to improving the curative effect of drugs. Experiments have proved that the selenium-enriched spirulina active peptides prepared by the present invention can be applied to the clinical treatment of ulcerative colitis, which is beneficial to reducing the treatment cost and side effects. Description of the Drawings

[0021] Figure 1 It is the influence of protease types on the hydrolysis degree and selenium recovery rate of selenium-enriched spirulina.

[0022] Figure 2 It is the influence of different solid-liquid ratios on the hydrolysis degree and selenium recovery rate of selenium-enriched spirulina.

[0023] Figure 3 It is the influence of enzyme dosage on the hydrolysis degree and selenium recovery rate of selenium-enriched spirulina.

[0024] Figure 4 It is the influence of enzymatic hydrolysis time on the hydrolysis degree and selenium recovery rate of selenium-enriched spirulina.

[0025] Figure 5 It is the influence of selenium-enriched spirulina peptides obtained by different preparation methods on the oxidative damage model of H2O2-induced Caco-2 cells.

[0026] Figure 6 It is the total ion current chromatogram of enzymatic hydrolysis of selenium-enriched spirulina peptides by the "one-step method" (a) and the "two-step method" (b).

[0027] Figure 7 It is the influence of selenium-enriched spirulina active peptides on the disease activity index of ulcerative colitis mice.

[0028] Figure 8 It is the influence of selenium-enriched spirulina active peptides on the colon length of ulcerative colitis mice.

[0029] Figure 9 It is the influence of selenium-enriched spirulina active peptides on the organ index of ulcerative colitis mice.

[0030] Figure 10 It is the influence of selenium-enriched spirulina active peptides on the colon cytokines (IL-1β, TNF-α) of ulcerative colitis mice.

[0031] Figure 11 It is the microscopic observation result of the distal colon HE section of ulcerative colitis mice treated with selenium-enriched spirulina active peptides.

[0032] Figure 12 It is the immunofluorescence microscopic observation result of the expression of ZO-1 and occludin proteins in the distal colon of ulcerative colitis mice treated with selenium-enriched spirulina active peptides.

[0033] Figure 13 Effect of selenium - rich spirulina active peptide on the expression levels of ZO - 1 and occludin proteins in ulcerative colitis mice Specific implementation manners

[0034] To better understand the present invention, the following further describes the present invention in detail with reference to embodiments and drawings for those skilled in the art to understand. The following embodiments do not limit the protection scope of the present invention, and any changes and variations made on the basis of the present invention are within the protection scope of the present invention

[0035] Embodiment 1

[0036] The selenium - rich spirulina peptide was prepared by the "one - step method". Selenium - rich spirulina and distilled water were mixed at a solid - liquid ratio of 1:40, then protease accounting for 4% of the mass of selenium - rich spirulina was added, and enzymatic hydrolysis was carried out at pH 8 and a temperature of 50°C for 2 hours. After heating to inactivate the enzyme and centrifuging, the supernatant was taken. Finally, the supernatant was separated by ultrafiltration membrane with a molecular weight cut - off of 5 kDa, and freeze - dried to obtain the peptide

[0037] This experiment investigated the effects of different types of proteases on the degree of hydrolysis and selenium recovery rate. The results are as Figure 1 shown. When alkaline protease was selected, the degree of hydrolysis of selenium - rich spirulina and the selenium recovery rate of the enzymatic hydrolysis product were the highest, reaching 15.18% and 27.44% respectively

[0038] Embodiment 2

[0039] The selenium - rich spirulina peptide was prepared by the "one - step method". Selenium - rich spirulina and distilled water were mixed at different solid - liquid ratios, then alkaline protease accounting for 3% of the mass of selenium - rich spirulina was added, and enzymatic hydrolysis was carried out at pH 9 and a temperature of 55°C for 2 hours. After heating to inactivate the enzyme and centrifuging, the supernatant was taken. Finally, the supernatant was separated by ultrafiltration membrane with a molecular weight cut - off of 5 kDa, and freeze - dried to obtain the peptide

[0040] This experiment investigated the effects of different solid - liquid ratios on the degree of hydrolysis and selenium recovery rate. The results are as Figure 2 shown. Different solid - liquid ratios had little effect on the degree of hydrolysis, but had a greater effect on the selenium recovery rate. When the solid - liquid ratio was 1:40, the selenium recovery rate was the highest (23.62%)

[0041] Embodiment 3

[0042] The selenium - rich spirulina peptide was prepared by the "one - step method". Selenium - rich spirulina and distilled water were mixed at a solid - liquid ratio of 1:40, then alkaline protease with different mass ratios of selenium - rich spirulina was added, and enzymatic hydrolysis was carried out at pH 10 and a temperature of 50°C for 2 hours. After heating to inactivate the enzyme and centrifuging, the supernatant was taken. Finally, the supernatant was separated by ultrafiltration membrane with a molecular weight cut - off of 5 kDa, and freeze - dried to obtain the peptide

[0043] This experiment investigated the effects of different enzyme addition amounts on the degree of hydrolysis and selenium recovery rate, and the results are as Figure 3 shown. The enzyme addition amount has a great influence on both the degree of hydrolysis and selenium recovery rate. When the enzyme addition amount is 4%, the degree of hydrolysis and selenium recovery rate are 16.11% and 21.38% respectively, and there is no significant difference compared with the addition of 5%.

[0044] Example 4

[0045] The "one-step method" was used to prepare selenium-enriched spirulina peptide. Selenium-enriched spirulina was mixed with distilled water at a solid-liquid ratio of 1:40, and then alkaline protease accounting for 4% of the mass of selenium-enriched spirulina was added. Enzymolysis was carried out at pH 8 and a temperature of 55 °C for 1 - 3 hours. After heating to inactivate the enzyme and centrifuging, the supernatant was taken. Finally, the supernatant was separated by ultrafiltration through a ultrafiltration membrane with a molecular weight cut-off of 5 kDa, and the peptide was obtained after freeze-drying.

[0046] This experiment investigated the effects of different enzymolysis times on the degree of hydrolysis and selenium recovery rate, and the results are as Figure 4 shown. The degree of hydrolysis and selenium recovery rate are the highest when enzymolysis is carried out for 2.5 hours, which are 16.10% and 27.23% respectively.

[0047] Test example

[0048] After comprehensive comparison, the optimal process for preparing selenium-enriched spirulina peptide by the "one-step method" was determined as follows: Selenium-enriched spirulina was mixed with distilled water at a solid-liquid ratio of 1:40, and then alkaline protease accounting for 4% of the mass of selenium-enriched spirulina was added. Enzymolysis was carried out at pH 8 and a temperature of 55 °C for 2.5 hours. After heating to inactivate the enzyme and centrifuging, the supernatant was taken. Finally, the supernatant was separated by ultrafiltration through a ultrafiltration membrane with a molecular weight cut-off of 5 kDa, and the peptide was obtained after freeze-drying.

[0049] At the same time, the traditional "two-step method", namely protein extraction - protease hydrolysis process, was used to prepare selenium-enriched spirulina peptide. The specific process is as follows: (1) Protein extraction: Selenium-enriched spirulina was first extracted with warm water, and the water extraction precipitate was then extracted with 0.05 mol / L sodium hydroxide solution to finally obtain selenium-enriched spirulina protein. (2) Enzymolysis: Selenium-enriched spirulina protein was mixed with distilled water at a solid-liquid ratio of 1:40, and then alkaline protease accounting for 4% of the mass of selenium-enriched spirulina protein was added. Enzymolysis was carried out at pH 8 and a temperature of 55 °C for 2 h. After heating to inactivate the enzyme and centrifuging, the supernatant was taken. (3) The supernatant was separated by ultrafiltration through a ultrafiltration membrane with a molecular weight cut-off of 5 kDa, and the peptide was obtained after freeze-drying.

[0050] 1. Determination of selenium content and degree of hydrolysis

[0051] The selenium content in selenium-enriched spirulina peptide was determined by hydride generation-atomic fluorescence spectrometry (HG-AFS), specifically referring to GB / T 21729-2008; the degree of hydrolysis of selenium-enriched spirulina was determined by the pH-stat method, as shown in Table 1.

[0052] The selenium content in the selenium peptide product prepared by the "one-step method" was 238.42 μg / g, higher than that of the product prepared by the "two-step method" (205.26 μg / g); the degree of protein hydrolysis obtained by the "one-step method" was 18.33%, higher than 12.81% of the "two-step method".

[0053] Table 1 Determination results of selenium content and degree of hydrolysis

[0054] Enzymatic hydrolysis method Degree of hydrolysis Selenium content Direct enzymatic hydrolysis 18.33％ 238.42 μg / g Protease hydrolysis 12.81％ 205.26 μg / g

[0055] 2. Determination of antioxidant capacity

[0056] The antioxidant effects of selenium-enriched spirulina peptides prepared by direct enzymatic hydrolysis of the "one-step method" and protease hydrolysis of the "two-step method" were verified using the H2O2-induced oxidative damage model of Caco-2 cells, as Figure 5 shown. When compared at the same dose concentrations of 100, 400, and 800 μg / mL (calculated as selenium), the SOD enzyme activity of the selenium peptides obtained by enzymatic hydrolysis of the "one-step method" was significantly higher than that of the "two-step method" (p<0.05).

[0057] 3. Structure identification of selenium-enriched spirulina peptide

[0058] The structure of selenium-enriched spirulina peptide was identified by liquid chromatography-mass spectrometry (UPLC-Q-Extractive Orbitrap MS). UPLC conditions: chromatographic column: Accucore C-18 (100×2.1 mm, 2.6 μm); mobile phase A: 0.1% formic acid-acetonitrile; B: 0.1% formic acid aqueous solution. Mass spectrometry conditions: ion source ESI+MS, spray voltage 3800 V, capillary temperature 275 °C. The liquid chromatography-mass spectrometry results were analyzed using the Qual Browser module in the Thermo Xcalibur 2.2 qualitative software. After preliminary analysis of the peptide sequence, the parsed peptide segments were searched and matched using the UniProt data (http: / / www.uniprot.org) to determine the structure of the peptide segments.

[0059] Figure 6 is the total ion chromatogram of selenium-enriched spirulina peptide.

[0060] The results are shown in Table 2 and Table 3. A total of 74 peptide segments containing 75 seleno - amino acids were obtained by the "two - step method", including 41 SeC and 34 SeM; a total of 80 peptide segments containing 80 seleno - amino acids were obtained by the "one - step method", including 43 SeC and 37 SeM. It is proved that compared with the traditional "two - step method" process, direct enzymatic hydrolysis by the "one - step method" can obtain selenium - rich spirulina peptides with a high selenium retention rate.

[0061] Table 2 Identification results of selenium - containing peptide segments in selenium - rich spirulina peptides enzymatically hydrolyzed by the "one - step method"

[0062]

[0063]

[0064] Table 3 Identification results of selenium - containing peptide segments in selenium - rich spirulina peptides enzymatically hydrolyzed by the "two - step method"

[0065]

[0066]

[0067] 4. Pharmacodynamic evaluation of selenium - rich spirulina peptides by the "one - step method" in the treatment of ulcerative colitis

[0068] After the mice were adapted to the environment for 3 days, they were randomly divided into 4 groups (n = 10). All groups were administered once a day by gavage (from day 0 to day 24): control group (Con, with distilled water), model group (Mod, with distilled water), low - dose SeSPs group (LSP, containing 10 μg / kg Se bw), and high - dose SeSPs group (HSP, containing 20 μg / kg Se bw). From the 14th day of modeling, except for the Con group, the Mod group, LSP group, and HSP group were given 2.5% DSS (dextran sulfate sodium salt) instead of normal drinking water. In addition, from the 15th day to the 24th day, the physical conditions of the mice, including body weight and fecal conditions, were recorded daily (experimental animal ethics approval number: HZAUMO - 2023 - 0220).

[0069] (1) Effects of selenium - rich spirulina peptides on DAI scores and organ indices of mice with ulcerative colitis

[0070] The severity of colitis in mice can be reflected by body weight, colon length, DAI score evaluation, and organ index. The supplementation of SeSPs significantly reduced the DAI score ( Figure 7 ), and significantly increased the colon length ( Figure 8 ). In addition, the liver index, spleen index, and kidney index of the Mod group were significantly higher than those of the Con group, and the thymus index was significantly lower than that of the Con group. After supplementing SeSPs, the organ indices of the mice tended to return to normal levels ( Figure 9)。Therefore, SeSPs can significantly improve colitis-related indicators.

[0071] (2) Effect of Se-enriched spirulina peptides on cytokines in the colon of mice

[0072] Cytokines (IL-1β, TNF-α) in the colon of mice were detected using an ELISA kit (Huding Biotechnology Co., Ltd., Shanghai, China). The results showed that the levels of inflammatory cytokines in the DSS treatment group were significantly increased, while the secretion levels of colonic inflammatory cytokines decreased significantly after the addition of Se-enriched spirulina peptides ( Figure 10 ).

[0073] (3) Effect of Se-enriched spirulina on the intestinal function barrier of mice with ulcerative colitis

[0074] Mouse colon tissues were fixed in 4% paraformaldehyde for more than 24 hours, then embedded in paraffin and serially sectioned (4 μm). The serial sections were stained with hematoxylin and eosin and photographed under an optical microscope, as Figure 11 shown.

[0075] Frozen sections (8 μm) of the distal colon of control animals and animals treated with DSS for 9 days (1 section per mouse) were fixed in 4% paraformaldehyde for 10 minutes. Then they were rinsed twice in PBS for 5 minutes each time, and then incubated with 200 μL of blocking buffer (10% donkey serum in PBS containing 0.1% Triton) in a humid chamber at 23 °C for 1 hour. Then they were incubated with occludin and ZO-1 antibodies overnight at 4 °C, and observed under a microscope after washing and counterstaining, as Figure 12 shown.

[0076] Through HE staining and immunofluorescence analysis of the colon tissues of mice with ulcerative colitis, the villi in the colon of mice after DSS treatment completely fell off, the mucosa was edematous and accompanied by a large number of inflammatory cell infiltrations, showing severe intestinal mucosal ulcers and erosions. After intervention with Se-enriched spirulina peptide SeSPs, the intestinal mucosal structure of mice remained intact, the number of goblet cells and intestinal villi returned to normal, and the expression intensities of tight junction (TJ) proteins such as ZO-1 and occludin could all return to normal levels ( Figure 13 ), so Se-enriched spirulina peptide SeSPs can repair the intestinal barrier function and maintain intestinal mucosal integrity.

Claims

1. A preparation method of selenium-enriched spirulina active peptides (SeSPs), characterized in that, One-step enzymatic hydrolysis method is adopted, which comprises the following steps: (a) Mixing selenium-enriched spirulina with water at a material-liquid mass ratio of 1:20 - 1:60; (b) Adding protease accounting for 2 - 6% of the mass of selenium-enriched spirulina, and carrying out enzymatic hydrolysis for 1 - 3 hours at pH 7.5 - 10 and a temperature of 45 - 60°C; (c) Inactivating the enzyme and then centrifuging to obtain the supernatant; (d) Filtering the supernatant through an ultrafiltration membrane with a molecular weight cut-off of 3 - 10 kDa, and obtaining selenium-enriched spirulina active peptides after freeze-drying.

2. The preparation method according to claim 1, wherein The protease is alkaline protease.

3. The preparation method according to claim 1, wherein The material-liquid ratio is 1:

40.

4. The preparation method according to claim 1, characterized in that, The enzymatic hydrolysis conditions are: pH 8 - 9, temperature 50 - 55°C, and enzymatic hydrolysis time of 2 - 2.5 hours.

5. The preparation method according to claim 1, characterized in that, The molecular weight cut-off of the ultrafiltration membrane is 5 kDa.

6. The preparation method according to claim 1, characterized in that, The enzyme addition amount is 4% of the mass of selenium-enriched spirulina.

7. A selenium-enriched spirulina active peptide prepared by the method according to any one of claims 1-6, characterized in that, The active peptide contains 80 selenoamino acids, including 43 selenocysteine (SeC) and 37 selenomethionine (SeM).

8. Use of the selenium-enriched spirulina active peptide according to claim 7 in the preparation of a drug for treating ulcerative colitis.

9. The application according to claim 8, wherein The active peptide exerts its function by regulating the intestinal oxidative stress pathway, inhibiting the secretion of inflammatory cytokines (IL-1β, TNF-α), and repairing the intestinal barrier function.

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