Preparation method of boletus edulis copper chelating peptide

Abstract

The invention discloses a preparation method of boletus edulis copper chelating peptide, which comprises the following steps: crushing boletus edulis sporocarp, sequentially carrying out enzymolysis and high-temperature enzyme deactivation on boletus edulis powder, and carrying out separation and purification by using gel chromatography by taking a copper chelating rate as a guide, so as to obtain and determine a component with the strongest copper chelating activity. Identifying the copper chelating peptide through nanoliter liquid chromatography-tandem mass spectrometry; according to the method for extracting, separating, purifying and identifying the boletus edulis copper chelating peptide, the structure and the copper chelating characteristic of the copper chelating peptide in the boletus edulis are shown, the novel copper chelating peptide is found in the boletus edulis for the first time, and the prepared copper chelating peptide has high copper chelating activity and can be used for preparing the copper chelating peptide. And copper lacking in the human body can be effectively supplemented, and good development prospects are achieved.

Description

Technical Field

[0001] The invention relates to the technical field of edible fungi deep processing, and in particular to a method for preparing a copper chelated peptide of Boletus lanmoides. Background Art

[0002] Copper is an essential trace element for humans. Its content in the human body ranks third, second only to iron and zinc. It is mainly distributed in muscles and bones, liver and blood. The copper content in adults is about 50-150 mg, and the copper content in normal human blood ranges from 10 to 24.6 μmol / L. Copper is the main component of many enzymes and certain proteins. Its main biochemical function is catalysis. Many copper-containing metalloenzymes are important oxidases that can reduce the content of molecular oxygen. Studies have shown that copper is a trace element necessary for infant growth, host defense mechanisms, bone strength maintenance, red blood cell and white blood cell maturation, iron transport, cholesterol and sugar metabolism. In addition, copper also plays some important non-enzymatic roles, such as angiogenesis, nerve myelin formation and endorphin activity. Lack of copper ions can lead to neurodegenerative diseases such as Alzheimer's disease and Parkinson's disease, idiopathic pulmonary fibrosis and diabetes.

[0003] After copper is ingested, the stomach absorbs only a small part of it, and the main absorption site is the duodenum. The absorption rate is affected by the dietary copper level, other nutrients such as vitamin C, and other mineral elements. 2+ Metal ions such as phytic acid and oxalic acid in the diet easily form precipitation, thereby reducing the bioavailability of copper. Therefore, even if there is more copper in the diet, the copper in the diet is mainly inorganic copper, and the bioavailability is affected by many factors. It is still necessary to prevent copper deficiency by taking copper supplements in addition to the diet.

[0004] Copper chelate peptides have good chemical stability, are easily absorbed by the intestine, and have high biological value. They can not only meet people's needs for amino acids, but also promote the metabolism of Cu. 2+ The absorption of peptides is mainly related to the human body's absorption mechanism of peptides. When the human body absorbs peptides, its main absorption mechanism is to enter the blood through the intestinal mucosa in a whole form. 2+ Compared with the absorption method, this absorption method is characterized by low energy consumption, fast absorption and not easy to saturate. This special transport mechanism avoids Cu 2+ It reacts with gastric acid, so it has little irritation to the gastrointestinal tract, and it also avoids reacting with substances such as phytic acid in the gastrointestinal tract, effectively improving the bioavailability of copper.

[0005] Yunnan Lanmao Boletus is rich in resources, rich in high-quality protein, vitamins and dietary fiber. The enzymatic hydrolysis of protein into polypeptides can combine with copper ions to convert inorganic copper into organic copper, and further absorb as a whole in the form of chelates with the help of the absorption and transport characteristics of polypeptides in the intestine. At the same time, the bioactive substances of Lanmao Boletus show biological activities such as antioxidant, anti-tumor, antibacterial and immunomodulatory. Using Lanmao Boletus as a protein base material source for the development of copper chelated peptides can not only make the pharmacological effects of bioactive peptides available, but also increase the added value of Lanmao Boletus products. Summary of the invention

[0006] The present invention provides a method for preparing a copper chelate peptide of Boletus lanmoides. The copper chelate peptide has good copper chelating activity and can be used to prepare a copper supplement, which can improve the quality of Cu 2+ Bioavailability in humans.

[0007] The present invention is achieved through the following technical solutions:

[0008] A method for preparing a copper chelated peptide of Boletus lanmoides, comprising the following specific steps:

[0009] (1) Processing of Boletus edulis fruiting bodies: select Boletus edulis fruiting bodies of uniform size and not fully opened, remove soil attachment, wash and dry with paper towels, cut into thin slices with a knife, dry to obtain dry matter, grind and sieve for later use; (2) Preparation of Boletus edulis enzymatic hydrolysate: mix the Boletus edulis powder prepared in step (1) with ultrapure water to prepare a suspension, adjust the pH to 7, add neutral protease, extract in a 50°C water bath with magnetic stirring for 3 h, place in a boiling water bath to inactivate the enzyme for 20 min, cool and centrifuge, take the supernatant, perform vacuum freeze drying, and collect and preserve the Boletus edulis crude polypeptide freeze-dried powder;

[0010] (3) Gel filtration chromatography separation: The crude lyophilized powder of Boletus lanmoides polypeptide is dissolved in ultrapure water to prepare a sample solution, which is then loaded into a chromatography column filled with Sephadex G-15 gel. The components corresponding to the peaks in the ultraviolet spectrum are collected and vacuum freeze-dried. The copper chelating activity is measured and the component with the strongest copper chelating activity is selected as the copper chelating peptide.

[0011] In step (1), the fruiting bodies of Boletus lanmoides are washed and dried at 30° C. for 5 h, then dried at 55° C. for 10 h, crushed and passed through a 60-mesh sieve.

[0012] In step (2), the ratio of the mixed liquid of Boletus edulis powder and ultrapure water is 1:25 (g / mL), 1 mol / L sodium hydroxide solution and 1 mol / L hydrochloric acid solution are used to adjust the pH value, the enzyme is inactivated in a 100°C boiling water bath for 20 min, and the centrifugation conditions are 4°C, 8000 r / min, and 20 min.

[0013] In step (3), the gel chromatography conditions are as follows: the concentration of the crude lyophilized powder of Boletus lanmoides polypeptide in the sample solution is 40 mg / mL, the flow rate is 1.0 mL / min, the sample volume is 1 mL, and the detection wavelength is 220 nm.

[0014] The present invention performs biological activity, toxicity and ADMET property analysis and prediction on copper chelating peptides according to PeptideRanker, ToxinPred and admetSAR 2.0 online websites, and simultaneously examines the polypeptide molecular weight (200-3000Da) and molecular docking binding energy. After the above screening, a copper chelating peptide HDIPL with the strongest copper chelating activity is obtained, and its amino acid sequence is: His-Asp-Ile-Pro-Leu.

[0015] Compared with the prior art, the present invention has the following beneficial effects:

[0016] The invention uses the fruiting body of Boletus lanmoides as a raw material, hydrolyzes the protein by enzymes, and then utilizes separation and purification technology and computer simulation technology to screen out a polypeptide sequence with the strongest copper chelating activity. The chelating rate of the chelating peptide is 86.66%.

[0017] The copper chelate peptide of Boletus lanmoides of the present invention has strong copper chelating activity and can improve the copper 2+ Bioavailability in humans allows for the preparation of copper supplements. BRIEF DESCRIPTION OF THE DRAWINGS

[0018] Figure 1 It is the chromatogram after Sephadex G-15 gel chromatography separation;

[0019] Figure 2 The gel components F1, F2 and Cu 2+ The chelation rate of

[0020] Figure 3 This is the mass spectrum of HDIPL. DETAILED DESCRIPTION

[0021] The present invention is further described below in conjunction with specific embodiments for better understanding, but the protection scope of the present invention is not limited thereto.

[0022] Example 1

[0023] A method for preparing a copper chelated peptide of Boletus lanmoides, comprising the following specific steps:

[0024] (1) Processing of Boletus edulis fruiting bodies: Select Boletus edulis fruiting bodies of uniform size and not fully opened, remove the dirt attached, wash and dry with a paper towel, cut into thin slices with a knife, place them on a tray, and use an electric hot air drying oven to dry them at 30° C. for 5 h, then increase the temperature to 55° C. and dry them for another 10 h. After drying, the dry matter is crushed and passed through a 60-mesh sieve for later use;

[0025] (2) Preparation of enzymatic hydrolysate of Boletus edulis: The Boletus edulis fruiting body powder prepared in step (1) was mixed with ultrapure water at a solid-liquid ratio of 1:25 (g / mL) to prepare a suspension, and the pH of the suspension was adjusted to 7 with 1 mol / L sodium hydroxide solution and 1 mol / L hydrochloric acid solution. Neutral protease (Dispase, CAS No.: 9068-59-1) was added at 6000 U / g of Boletus edulis fruiting body powder, and the mixture was placed in a 50°C water bath with magnetic stirring for 3 h. After the extraction, the mixture was placed in a 100°C boiling water bath for 20 min to inactivate the enzyme. After cooling, the mixture was centrifuged at 4°C and 8000 r / min for 20 min, and the supernatant was vacuum freeze-dried at -55°C for 48 h. The obtained crude polypeptide freeze-dried powder of Boletus edulis was stored in a -80°C refrigerator;

[0026] (3) Gel filtration chromatography separation: The crude lyophilized powder of Boletus lanmoides polypeptide was dissolved in ultrapure water to prepare a 40 mg / mL sample solution. After filtering the sample solution using a 0.45 μm aqueous phase filter membrane, 1 mL of the sample solution was loaded into a Sephadex G-15 gel chromatography column. At 25±5°C, the UV detector absorption wavelength was adjusted to 220 nm, and ultrapure water was used as the eluent at a flow rate of 1 mL / min. The components corresponding to each peak in the UV spectrum were collected, combined and freeze-dried, and then stored in a refrigerator at -80°C. Figure 1 It can be seen that two components were separated by gel chromatography: F1 and F2.

[0027] Determination of copper chelating activity: prepare 30 mg / mL sample solution, take 100uL and add 100uL of 1mg / mL copper sulfate solution, add 2.8mL sodium acetate buffer (pH=6, 50mM) and 60uL of 4mM catechol violet solution, mix well and react at room temperature for 30min, measure its absorbance at 632nm wavelength, use water as blank control and EDTA as positive control:

[0028]

[0029] Depend on Figure 2 It can be seen that among the two fractions obtained by gel filtration chromatography, the copper chelating activity of F1 is higher than that of F2 and is close to that of EDTA. Based on this result, F1 was selected as the target fraction.

[0030] (4) Sequence identification of copper chelating peptides

[0031] Nano-LC-MS / MS was used to sequence and identify the copper chelating peptide components separated by gel chromatography:

[0032] 1) Sample treatment: Take an appropriate amount of sample and dissolve it in 50mM NH4HCO3 solution, then add DTT solution to make the final concentration of 10mmol / L, reduce it in a 56℃ water bath for 1h, then add IAM solution to make the final concentration of 55mmol / L, react in the dark for 40min, and use self-filled desalting C 18 The column was desalted, the solvent was evaporated in a vacuum centrifugal concentrator at 45°C, and the sample was tested for later use.

[0033] 2) Liquid phase conditions were as follows: Reprosil-Pur 120C18-AQ chromatographic column (100 μm id×180 mm, 3 μm); mobile phase A was 0.1% formic acid in water, and mobile phase B was 0.1% formic acid in water and 80% acetonitrile; flow rate was 600 nL / min, gradient elution: 0-2 min, 4%-8% B; 2-35 min, 8%-28% B; 35-55 min, 28-40% B; 55-56 min, 40-95% B; 56-66 min, 95% B.

[0034] 3) Mass spectrometry conditions were as follows: ESI+ ionization mode, full scan acquisition (m / z 100-1500), spray voltage 2.2KV, capillary temperature 270°C, primary mass spectrometry parameter settings: scan range 100-1500m / z, resolution 70000, maximum scan fill time 60ms, AGC target 3e6; secondary mass spectrometry parameter settings: resolution 17500, maximum scan fill time 50ms, AGC target 1e5; the raw files of mass spectrometry acquisition were searched through the Byonic database software to obtain the peptide sequence identification results.

[0035] (5) Peptide sequence screening

[0036] 1) Molecular weight screening: Molecular weight is 200-3000Da, which has strong chelating activity, so the peptides of 200-3000Da are selected.

[0037] The molecular weights of the polypeptides in the F1 fraction are shown in Table 1 ;

[0038] Table 1

[0039]

[0040] 2) Biological activity prediction: The biological activity of the chelated peptides was predicted according to PeptideRanker, and the activity was greater than 0.5. The biological activity prediction of the peptides in the F1 component is shown in Table 2;

[0041] Table 2

[0042]

[0043] 3) Prediction of potential toxicity and allergenicity: ToxinPred was used to predict the toxicity of the identified peptides, and it was found that all peptides were non-toxic and non-allergenic.

[0044] 4) ADMET property analysis: The peptide sequences were converted into SMILES format using the PepSMI tool, and then the absorption, distribution, metabolism, excretion and toxicity (ADMET) properties and water solubility of these active peptides were predicted on the admetSAR 2.0 website. The indicators selected were human intestinal absorption characteristics, acute oral toxicity, blood-brain barrier permeability and water solubility. It was found that 17 peptides had good water solubility, intestinal absorption characteristics, blood-brain barrier permeability and low oral toxicity.

[0045] 5) Molecular docking: The peptide sequence was converted into SMILES format using the PepSMI tool, and the two-dimensional structure was obtained by Chemdraw software, which was converted into a three-dimensional structure by Chem3D and adjusted to energy minimization using the minimization algorithm. The structures of ferrous ions and cupric ions were downloaded from Pubchem, and docking simulation was performed using AutoDock (1.5.7). Finally, pymol was used to visualize the molecular docking results. The 17 peptides obtained from the above screening in the F1 component were compared with Cu 2+ The molecular docking binding energy is shown in Table 4.

[0046] Table 4

[0047]

[0048] The potential copper chelating peptides screened based on the above five strategies are sorted out for further research.

[0049] (6) Synthesis of copper chelating peptides and verification of copper chelating activity:

[0050] Suzhou Modifu Biotechnology Co., Ltd. was commissioned to synthesize the identified HDIPL peptide using the Fmoc solid phase method. Its mass spectrum is shown below. Figure 3 As shown, the purity of the synthesis is more than 95%. The sample was dissolved to prepare a solution with a concentration of 10 mg / mL, and the copper chelating activity was verified. It was found that the copper chelating rate was 86.66%.

[0051] The specific implementation modes of the present invention are described in detail above in conjunction with the accompanying drawings, but the present invention is not limited to the above implementation modes, and various changes can be made within the knowledge scope of ordinary technicians in this field without departing from the purpose of the present invention.

Claims

1. A method for preparing a copper-chelated peptide of Boletus lanmoides, characterized in that: The specific steps are as follows: (1) Processing of Boletus edulis fruiting bodies: removing soil from Boletus edulis fruiting bodies, washing and drying, cutting into thin slices, drying to obtain dry matter, and crushing and sieving for later use; (2) Preparation of Boletus edulis enzymatic hydrolysate: The Boletus edulis powder prepared in step (1) is mixed with ultrapure water to form a suspension, the pH is adjusted to 7, neutral protease is added, and extraction is carried out in a 50° C. water bath with magnetic stirring for 3 h. After the extraction, the suspension is placed in a boiling water bath for 20 min to inactivate the enzyme, cooled and centrifuged, the supernatant is taken, and vacuum freeze-dried to collect and store the Boletus edulis crude polypeptide freeze-dried powder; (3) Gel filtration chromatography separation: The crude lyophilized powder of Boletus lanmoides polypeptide is dissolved in ultrapure water to prepare a sample solution, which is then loaded into a chromatography column filled with Sephadex G-15 gel. The components corresponding to the peaks in the ultraviolet spectrum are collected and vacuum freeze-dried. The copper chelating activity is measured and the component with the strongest copper chelating activity is selected as the copper chelating peptide.

2. The preparation method according to claim 1, characterized in that: In step (1), the fruiting bodies of Boletus lanmoides are washed and dried at 30° C. for 5 h, then dried at 55° C. for 10 h, crushed and passed through a 60-mesh sieve.

3. The preparation method according to claim 1, characterized in that: In step (2), the ratio of the mixed liquid of Boletus edulis powder and ultrapure water is 1:25 g / mL, 1 mol / L sodium hydroxide solution and 1 mol / L hydrochloric acid are used to adjust the pH value, the boiling water bath temperature is 100°C, and the centrifugal conditions are 4°C, 8000 r / min, and 20 min.

4. The preparation method according to claim 1, characterized in that: In step (3), the gel chromatography conditions are as follows: the concentration of the crude lyophilized powder of Boletus lanmoides polypeptide in the sample solution is 40 mg / mL, the flow rate is 1.0 mL / min, the sample volume is 1 mL, and the detection wavelength is 220 nm.

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