Lucid ganoderma sporoderm-broken spore powder antioxidant protein peptide and preparation method thereof

Abstract

The invention relates to a ganoderma lucidum wall-broken spore powder antioxidant protein peptide and a preparation method thereof. The amino acid sequence of the antioxidative protein peptide of the ganoderma lucidum sporoderm-broken spore powder disclosed by the invention is as shown in SEQ. ID. NO.1-Phe-Ala-Asp-Glu-Asn-Phe-Lys. The preparation method comprises the following steps: (1) raw material pretreatment: taking residual residues obtained after processing of ganoderma lucidum sporoderm-broken spore powder as raw materials, and adding chitinase with the enzyme amount of 2000-4000 U / g into the raw materials according to a solid-liquid ratio of 1: 20 (g / mL); (2) extracting protein: mixing the filter residue and deionized water according to a material-liquid ratio of (1: 10)-(1: 20) (g / mL); (3) biological enzymolysis: trypsin is adopted, the enzyme addition amount is 6000-9000 U / g, the enzymolysis time is 2-4 hours, the enzymolysis temperature is 40-60 DEG C, and the pH value is 7.5-8.5; (4) ultrafiltration membrane separation: separating the enzymatic hydrolysate by using an ultrafiltration membrane with the molecular weight cutoff of 3KD; and (5) continuous chromatographic separation: separating the crude peptide enzymatic hydrolysate by using a sephadex chromatographic column to prepare the ganoderma lucidum wall-broken spore powder antioxidant protein peptide. The protein resource utilization degree of the ganoderma lucidum sporoderm-broken spore powder residues is improved.

Description

TECHNICAL FIELD

[0001] The present application relates to the technical field of preparation of antioxidant polypeptides, and particularly relates to a ganoderma lucidum broken spore powder antioxidant protein peptide and a preparation method thereof. BACKGROUND

[0002] Excessive free radicals in the human body can cause oxidative stress, thereby destroying the original redox balance of the body, causing a variety of chronic diseases such as arteriosclerosis, cardiovascular disease, neurodegenerative disease, cancer and aging. Therefore, it is crucial to eliminate excess free radicals in the body and maintain the balance of the human body homeostasis for preventing and treating the occurrence of related diseases. Natural antioxidants, as excellent hydrogen donors, have a significant free radical scavenging effect, are quickly absorbed in the human body, have good effects, and are non-toxic and have no side effects. Therefore, in recent years, its demand in the functional food and drug market has also increased day by day.

[0003] Natural antioxidant active polypeptides are usually composed of an amino acid sequence of 2-20 amino acids. They are favored by researchers due to their characteristics of wide sources, significant activity and high safety, are one of the most popular research topics in the international food industry, and are functional factors with great development prospects. At present, the proteins used for preparing antioxidant polypeptides are mostly derived from animal proteins and plant proteins, and the development of fungal proteins is relatively backward, especially the reports on ganoderma lucidum proteins are few. Most of the reported functional polypeptides derived from ganoderma lucidum belong to glycopeptides, and the active units are mainly glycoside units, without indicating the function of the polypeptide segment in the substance.

[0004] Ganoderma spore powder, as the reproductive cell of ganoderma lucidum, is an extremely fine particle ejected from the cap of ganoderma lucidum in the late growth and maturation stage of ganoderma lucidum. It contains polysaccharides, triterpenes, proteins, nucleotides and other pharmacologically active ingredients, and has outstanding performance in alcoholism prevention and liver protection, antioxidant, antitumor, immunomodulation, antibacterial, anti-aging and other aspects. Among them, active polysaccharides and triterpenes have been successfully developed into functional foods, while protein substances have been severely underestimated as a high-quality protein resource for various bioactive peptides. Therefore, the research on the development of bioactive peptides from ganoderma lucidum protein resources by modern biotechnology has broad prospects.

[0005] Enzymatic hydrolysis is one of the important methods for preparing polypeptides from proteins. The activity of the enzymatic hydrolysis product is limited by the protein raw material and enzyme preparation. When different enzyme preparations act on proteins, the specificity of the enzyme cleavage site will lead to different types of amino acids and peptide segment structures at the carboxyl and amino terminals of the enzymatic hydrolysis product.

[0006] The existing ganoderma lucidum spore powder still contains about 18% of protein after being extracted by spore oil, polysaccharide, triterpene and the like, but most of the residues after processing are directly discarded as waste or made into spore particles with low value, causing great waste of resources. At present, there is no method for preparing ganoderma lucidum spore powder antioxidant protein peptide by using the residues after processing of broken ganoderma lucidum spores as raw materials. SUMMARY

[0007] The present application aims to overcome the above-mentioned deficiencies in the prior art and provide a ganoderma lucidum spore powder antioxidant protein peptide and a preparation method thereof, which can improve the utilization degree of protein resources of broken ganoderma lucidum spore powder residues.

[0008] The technical scheme adopted by the present application to solve the above-mentioned problems is as follows: a ganoderma lucidum spore powder antioxidant protein peptide, characterized in that the amino acid sequence thereof is SEQ.ID.NO.1-Phe-Ala-Asp-Glu-Asn-Phe-Lys.

[0009] A preparation method of a ganoderma lucidum spore powder antioxidant protein peptide, characterized by the following steps:

[0010] (1) Raw material pretreatment: using the residues after processing of broken ganoderma lucidum spores as raw materials, the raw materials are treated with chitinase at an enzyme amount of 2000-4000 U / g under the extraction conditions of 45°C and pH value of 6.0-8.0 at a solid-liquid ratio of 1:20 (g / mL) for 2-3 h, and then the enzyme is inactivated by boiling water bath for 10 min; hot filtration is performed, and the filter residue is reserved;

[0011] (2) Protein extraction: the filter residue is mixed with deionized water at a solid-liquid ratio of 1:10-1:20 (g / mL), the pH value is adjusted to 8.5-10.5 by using 1 mol / L NaOH, the extraction time is 2-4 h, the temperature is 40-60°C, and after centrifugation at 8000 r / min for 15 min, the supernatant is collected, concentrated to 1 / 3 of the volume of the supernatant by rotary evaporation at 50°C, 6% (V / V) H2O2 is added for 3 h, the pH value is adjusted to the isoelectric point of 2.5-4 by using 0.1 mol / L HCl solution, and the precipitated protein is collected, washed with deionized water until neutral, and freeze-dried;

[0012] (3) Biological enzymolysis: trypsin is used, the protein is mixed with deionized water at a solid-liquid ratio of 1:10-1:30 (g / mL), the enzyme amount is 6000-9000 U / g, the enzymolysis time is 2-4 h, the enzymolysis temperature is 40-60°C, the pH value is 7.5-8.5, after the enzymolysis reaction is completed, the enzyme is inactivated at 100°C for 10 min to terminate the reaction, and after cooling to room temperature, centrifugation is performed at 8000 r / min for 15 min, the supernatant is collected, and the pH value is adjusted to 7.0 to obtain an enzymolysis liquid for standby use.

[0013] (4) Ultrafiltration membrane separation: the enzyme solution is separated by using an ultrafiltration membrane with a molecular weight cut-off of 3KD, and a crude peptide enzyme solution with a molecular weight of ≤3kDa is collected;

[0014] (5) Continuous chromatographic separation: the crude peptide enzyme solution is separated by a dextran gel chromatography column, and ABTS + with the highest free radical scavenging rate is subjected to further separation by elution, and then purified by using a reverse C18 chromatography column, with each 1mL as a tube, and the ABTS + with the highest free radical scavenging rate is collected after freeze-drying, to obtain the ganoderma broken-spore powder antioxidant protein peptide, and the amino acid sequence is SEQ.ID.NO.1-Phe-Ala-Asp-Glu-Asn-Phe-Lys.

[0015] Preferably, the parameters for the ultrafiltration membrane separation and purification in step (4) of the present application are as follows: a pressure difference of 0.1-0.5MPa, a degradation liquid temperature of 15-50℃, a membrane area of 1.5-5m 2 , and an enzyme solution membrane flux of 0.1-2.5m 2 / h.

[0016] Preferably, the parameters for the ultrafiltration membrane separation and purification in step (4) of the present application are as follows: a pressure difference of 0.15-0.3MPa, a degradation liquid temperature of 20-30℃, a membrane area of 1.5-2.5m 2 , and an enzyme solution membrane flux of 0.2-0.8m 2 / h.

[0017] Preferably, the dextran gel in step (5) of the present application is a Sephadex G-25 gel chromatography column (XK 16 / 70mm, Pharmacia), with an elution flow rate of 0.5mL / min, and an ultraviolet detection wavelength of OD280nm, and each 2mL as a tube.

[0018] Preferably, the reverse C18 chromatography column in step (5) of the present application is a SunFire Prep C18 OBD TM (19mm×250mm×5μm, Waters) chromatography column, and the preparation separation conditions are as follows: mobile phase A: 0.1% trifluoroacetic acid aqueous solution; mobile phase B: 0.1% trifluoroacetic acid methanol solution; gradient elution program: gradient elution: 0-15min, 30-40% B; 15-35min, 40-90% B, injection amount: 500μL; elution flow rate: 1mL / min, column temperature: 30℃, and each fraction is detected and collected at a detection wavelength of 280nm.

[0019] A method for identifying the amino acid sequence of ganoderma lucidum spore powder antioxidant protein peptide, characterized by the following steps: using nano-LC-MS / MS high performance liquid chromatography-mass spectrometry to identify ABTS + The active polypeptide with the best free radical scavenging rate is identified by MAIDL-TOF mass spectrometry, the secondary mass spectrum is obtained by ESI-MS / MS, and the amino acid sequence of the high-activity peptide is identified by denovo sequencing.

[0020] A method for evaluating the in-vitro biological activity of ganoderma lucidum spore powder antioxidant protein peptide, characterized by the following steps: using H2O2-induced in-vitro liver cancer cell HepG2 oxidative stress model to further verify the screened active polypeptide component, detecting its activity on related antioxidant enzymes such as ROS, MDA, GSH, GSSG in cells, and the expression influence of Keap1-Nrf2 signal pathway related proteins. The polypeptide molecule can effectively prevent H2O2-induced liver cancer cell HepG2 membrane damage, reduce the production of ROS in oxidative damage cells, reduce the generation of MDA, increase the activity of antioxidant enzymes CAT, SOD, GSH-Px and the ratio of GSH / GSSG, can effectively occupy the binding site of Nrf2 and Keap1 through the Keap1-Nrf2 signal pathway, promote the effective dissociation of Nrf2 and Keap1, promote Nrf2 to enter the nucleus to regulate the transcription and expression of related antioxidant genes such as HO-1, thereby reducing H2O2-induced oxidative damage.

[0021] Compared with the prior art, the present application has the following advantages and effects: taking the signal pathway Keap1-Nrf2 which plays a key defense role in oxidative stress damage of the body as the target, according to the characteristics of ganoderma lucidum spore protein, a series of directional processing methods such as suitable biological enzymes, ultrafiltration, chromatographic separation are adopted, Nrf2 factor is effectively released by inhibiting the activity of Keap1 enzyme, and a ganoderma lucidum spore powder active protein peptide with strong antioxidant effect is successfully prepared. A new sequence of natural polypeptide antioxidant SEQ.ID.NO.1-Phe-Ala-Asp-Glu-Asn-Phe-Lys is prepared from the remaining material of ganoderma lucidum spore powder, and the preparation method is simple and easy to operate. The present application provides a new idea for the re-development of ganoderma lucidum spore powder protein resources, and has far-reaching significance for the development of health products, medicine and food industry. BRIEF DESCRIPTION OF DRAWINGS

[0022] In order to more clearly illustrate the technical solutions in the embodiments of the present application and / or the prior art, the following will briefly introduce the drawings needed to be used in the embodiments and / or prior art description.

[0023] Figure 1(a): gel chromatography separation elution peak curve of ganoderma lucidum spore powder protein crude peptide in Example 1;

[0024] Figure 1(b): DPPH radical scavenging rate and ABTS of different polypeptide fractions separated by Sephadex G-25 gel in Example 1 + • Radical scavenging rate bar chart;

[0025] Figure 2(a): RP-HPLC elution peak curve of antioxidant peptide P3-GLSP in Example 1;

[0026] Figure 2(b): is the corresponding antioxidant result bar chart of Figure 2(a);

[0027] Figure 3 : Secondary fragment ion map of polypeptide sequence FADENFK;

[0028] Figure 4 : 2D map of molecular docking of antioxidant protein peptide FADENFK and Keap 1;

[0029] Figure 5(a): RMSD kinetic result map of FADENFK-Keap 1 complex and Keap 1 protein;

[0030] Figure 5(b): RMSF kinetic result map of FADENFK-Keap 1 complex and Keap 1 protein;

[0031] Figure 5(c): Rg kinetic result map of FADENFK-Keap 1 complex and Keap 1 protein;

[0032] Figure 5(d): SASA kinetic result map of FADENFK-Keap 1 complex and Keap 1 protein;

[0033] Figure 5(e): Number of hydrogen bonds kinetic result map of FADENFK-Keap 1 complex and Keap 1 protein;

[0034] Figure 6(a): Fluorescence map of the effect on ROS level of HepG2 cells in the control group;

[0035] Figure 6(b): Fluorescence map of the effect on ROS level of HepG2 cells in the model group;

[0036] Figure 6(c): Fluorescence map of the effect on ROS level of HepG2 cells in the 12.5 μg / mL polypeptide group;

[0037] Figure 6(d): Fluorescence map of the effect on ROS level of HepG2 cells in the 25 μg / mL polypeptide group;

[0038] Figure 6(e): Fluorescence map of the effect on ROS level of HepG2 cells in the 50 μg / mL polypeptide group;

[0039] Figure 6(f): fluorescence chart of the effect of polypeptide on ROS level of HepG2 cells in 100 μg / mL polypeptide group;

[0040] Figure 6(g): column chart of the effect of polypeptide on ROS level of HepG2 cells under different polypeptide concentrations;

[0041] Figure 7(a): Western blotting strip chart of the expression of Keap1-Nrf2 pathway related proteins, wherein A is a control group; B is a 12.5 μg / mL polypeptide group; C is a 25 μg / mL polypeptide group; D is a 50 μg / mL polypeptide group; and E is a 100 μg / mL polypeptide group;

[0042] Figure 7(b): quantitative chart of the phosphorylation level of Keap1-Nrf2 pathway related proteins, wherein the polypeptide group concentrations are 12.5 μg / mL, 25 μg / mL, 50 μg / mL and 100 μg / mL in turn, and letters a to d represent significant differences from the control group. DETAILED DESCRIPTION

[0043] The present application will be further described in detail below with reference to the accompanying drawings and by examples, and the following examples are used to explain the present application but the present application is not limited to the following examples.

[0044] Example 1:

[0045] The preparation method of the ganoderma lucidum spore powder antioxidant protein peptide in the present embodiment has the following steps:

[0046] (1) raw material pretreatment: the ganoderma lucidum spore powder residue is treated with 2000 U / g of chitinase enzyme amount (Nanning Pangbo Biological Engineering, 20000 U / g) at a solid-liquid ratio of 1:20 (g / mL), 45°C and pH 8.0 for 3h, and then boiled in water bath for 10 min to inactivate the enzyme. Hot filtration is performed, and the filter residue is reserved.

[0047] (2) protein extraction: the filter residue is mixed with deionized water at a solid-liquid ratio of 1:10 (g / mL), the pH is adjusted to 8.5 with 1 mol / L NaOH, the extraction time is 2-4h, the temperature is 40-60°C, and the supernatant is collected after centrifugation at 8000 r / min for 15 min. The supernatant is concentrated to 1 / 3 of the original volume by rotary evaporation at 50°C, 6% (V / V) H2O2 is added for 3h, the pH value is adjusted to the isoelectric point 2.5 with 0.1 mol / L HCl solution, and the precipitated protein is washed with deionized water to neutral, and then freeze-dried.

[0048] (3) Bio-enzymatic hydrolysis: Trypsin (Nanning Pangbo Bioengineering, 4000U / g) was used. The protein and deionized water were mixed at a ratio of 1:10g / mL, the enzyme addition was 6000U / g, the hydrolysis time was 4h, the hydrolysis temperature was 40℃, and the pH was 7.5. After the hydrolysis reaction was completed, the enzyme was inactivated at 100℃ for 10min to stop the reaction. After cooling to room temperature, the mixture was centrifuged at 8000r / min for 15min. The supernatant was collected and the pH was adjusted to 7.0 to obtain the hydrolysate.

[0049] (4) Ultrafiltration membrane separation: Use an ultrafiltration membrane with a molecular weight cutoff of 3KD to separate the enzymatic hydrolysate and collect crude peptide enzymatic hydrolysate with a molecular weight ≤3kDa.

[0050] (5) Continuous chromatographic separation: Continuous chromatographic separation: using... The purification system used a Sephadex G-25 (XK 16 / 70 column) gel filtration column. The optimal antioxidant component in the crude peptide hydrolysate was filtered through a 0.45 μm microporous membrane. 0.15 M sodium chloride was used as the eluent, and the elution flow rate was 0.5 mL / min. The UV detection wavelength was OD 280 nm. Each 2 mL tube was connected to a elution tube. The number of tubes was combined according to the number of elution peaks. After the elution peaks were concentrated and lyophilized, they were reconstituted and adjusted to a uniform concentration of 0.5 mg / mL for antioxidant activity determination.

[0051] The elution time of the ultrafiltration components inside the gel column is as follows: Figures 1(a) to 1(b) As shown in the elution curve, three peaks were observed, yielding three fractions with different molecular weights. The free radical scavenging rate of the fractions increased as the molecular weight decreased from P1 to P3. At a concentration of 0.5 mg / ml, P3 exhibited the best antioxidant effect, followed by DPPH and ABTS. + The free radical scavenging rates reached 87.18% and 96.00%, respectively. The elution curves showed that the fraction eluted earlier had the smallest molecular weight, consistent with the rule that smaller peptides have higher antioxidant activity. Furthermore, the proportions of the three separated fractions with molecular weights of 2000-3000 Da, 2000-1500 Da, and less than 1500 Da were 10.48±0.62%, 18.61±0.58%, and 23.67±0.52%, respectively, while most peptides in fraction P3 were below 1500 Da.

[0052] The highly active component P3, rated as Sephadex G-25, was selected and analyzed using a reverse-phase C18 column with SunFire Prep C18OBD. TMA chromatographic column (19mm × 250mm, 5μm, Waters) was used to further separate and purify the fraction with the strongest antioxidant activity. The preparative separation conditions were as follows: Mobile phase A: 0.1% aqueous solution of trifluoroacetic acid; Mobile phase B: 0.1% methanol solution of trifluoroacetic acid; Gradient elution program: Gradient elution: 0–15 min, 30–40% B; 15–35 min, 40–90% B; Injection volume: 500 μL; Elution flow rate: 1 mL / min; Column temperature: 30℃; UV detection wavelength: OD 280 nm. Each fraction was collected, and its DPPH and ABTS were determined. + • Free radical scavenging activity. For example... Figures 2(a) to 2(b) As shown, the absorbance values ​​of each peak are very low, which is due to the small sample volume. Several small impurity peaks appear in the first 8 minutes of elution, all below 50 mAU, and can be ignored. Among them, a high solvent peak appears at 2.23 minutes, which is not included in the results. A single peak appears during the elution time of 18-35 minutes. After freeze-drying, this fraction is collected as P3-GLSP. After reconstitution, it is prepared at 0.5 mg / mL to determine the antioxidant activity. It was found that the ABTS after purification was... + Both the activity and DPPH activity were higher than the antioxidant activity of the unpurified component P3.

[0053] Example 2:

[0054] The steps for preparing the antioxidant protein peptides from Ganoderma lucidum spore powder in this embodiment are as follows:

[0055] (1) Raw material pretreatment: The residue of Ganoderma lucidum spore powder was treated with chitinase (Nanning Pangbo Biotechnology Co., Ltd., 20000 U / g) at a material-to-liquid ratio of 1:20 (g / mL) for 3 hours at 45℃ and pH adjusted to 7.0. After the reaction was completed, the enzyme was inactivated by boiling in a water bath for 10 minutes. The mixture was filtered while hot, and the filter residue was retained.

[0056] (2) Protein extraction: The filter residue and deionized water were mixed at a ratio of 1:20 (g / mL). The pH was adjusted to 10.5 with 1 mol / L NaOH. The extraction time was 3 h and the temperature was 60℃. After centrifugation at 8000 r / min for 15 min, the supernatant was collected. The supernatant was concentrated to 1 / 3 of the volume of the supernatant by rotary evaporation at 50℃. 6% H2O2 (V / V) was added for decolorization for 3 h. The pH was adjusted to the isoelectric point 4 with 0.1 mol / L HCl solution. The precipitated protein was collected, washed with deionized water several times until neutral, and then freeze-dried.

[0057] (3) Bio-enzymatic hydrolysis: Trypsin (Nanning Pangbo Bioengineering, 4000U / g) was used. The protein and deionized water were mixed at a ratio of 1:30 (g / mL). The enzyme addition was 9000U / g. The hydrolysis time was 2h, the hydrolysis temperature was 60℃, and the pH was 8.5. After the hydrolysis reaction was completed, the enzyme was inactivated at 100℃ for 10min to stop the reaction. After cooling to room temperature, the mixture was centrifuged at 8000r / min for 15min. The supernatant was collected and the pH was adjusted to 7.0 to obtain the hydrolysate.

[0058] (4) Ultrafiltration membrane separation: Use an ultrafiltration membrane with a molecular weight cutoff of 3KD to separate the enzymatic hydrolysate, collect crude peptide enzymatic hydrolysate with a molecular weight ≤3kDa, and concentrate it under vacuum to 1 / 3 of the original volume.

[0059] (5) Continuous chromatographic separation: Continuous chromatographic separation: using... The purification system used a Sephadex G-25 (XK 16 / 70 column) gel filtration column. The optimal antioxidant component was filtered through a 0.45 μm microporous membrane. 0.15 M sodium chloride was used as the eluent, and the elution flow rate was 0.5 mL / min. The UV detection wavelength was OD280 nm. Each 2 mL tube was connected to a elution tube. The number of tubes was combined according to the number of elution peaks, and the elution peaks were concentrated and lyophilized.

[0060] For the highly active component P3, the reaction was performed using a reverse C18 column, SunFire Prep C18 OBD. TM A chromatographic column (19mm × 250mm, 5μm, Waters) was used to further separate and purify the fraction with the strongest antioxidant activity. The preparative separation conditions were as follows: Mobile phase A: 0.1% aqueous solution of trifluoroacetic acid; Mobile phase B: 0.1% methanol solution of trifluoroacetic acid; Gradient elution program: Gradient elution: 0–15 min, 30–40% B; 15–35 min, 40–90% B; Injection volume: 500 μL; Elution flow rate: 1 mL / min; Column temperature: 30℃; UV detection wavelength: OD 280 nm. Each fraction was collected, and its DPPH and ABTS were determined. + • Free radical scavenging activity. The component with the highest free radical scavenging rate was collected after freeze-drying, which is the antioxidant protein peptide of Ganoderma lucidum spore powder with an amino acid sequence of SEQ.ID.NO.1-Phe-Ala-Asp-Glu-Asn-Phe-Lys, which is FADENNFK in single letter form.

[0061] Example 3:

[0062] The steps for preparing the antioxidant protein peptides from Ganoderma lucidum spore powder in this embodiment are as follows:

[0063] (1) Raw material pretreatment: Ganoderma lucidum spore powder residue was treated with 4000 U / g of cellulase enzyme (Nanning Pobio Bioengineering, 20000 U / g) at a solid-liquid ratio of 1:20 (g / mL) at 45°C and pH 6.0 for 3 h. After the reaction was completed, the enzyme was inactivated by boiling in a water bath for 10 min. Hot filtration was performed, and the filter residue was retained.

[0064] (2) Protein extraction: The filter residue was mixed with deionized water at a solid-liquid ratio of 1:15 (g / mL), and the pH was adjusted to 9.5 with 1 mol / L NaOH. The extraction time was 3 h at 50°C, and the supernatant was collected after centrifugation at 8000 r / min for 15 min. The supernatant was concentrated to 1 / 3 of the original volume by rotary evaporation at 50°C. 6% H2O2 (V / V) was added for 3 h of decolorization. The pH was adjusted to the isoelectric point of 3.5 with 0.1 mol / L HCl solution. The precipitated protein was washed with deionized water until it was neutral. It was then freeze-dried.

[0065] (3) Biological enzymatic hydrolysis: Trypsin (Nanning Pobio Bioengineering, 4000 U / g) was used to hydrolyze the protein with deionized water at a solid-liquid ratio of 1:20 (g / mL) and an enzyme dosage of 8000 U / g. The enzyme hydrolysis time was 3 h at 50°C and the pH was 8.0. After the enzyme hydrolysis reaction was completed, the enzyme was inactivated at 100°C for 10 min to terminate the reaction. After cooling to room temperature, the mixture was centrifuged at 8000 r / min for 15 min. The supernatant was collected and the pH was adjusted to 7.0 to obtain the enzyme hydrolysate.

[0066] (4) Ultrafiltration membrane separation: The enzyme hydrolysate was separated using a 3KD molecular weight cut-off ultrafiltration membrane. The crude peptide hydrolysate with a molecular weight of ≤3 kDa was collected and vacuum concentrated to 1 / 3 of the original volume.

[0067] (5) Continuous chromatography separation: The purified system used a Sephadex G-25 (XK16 / 70 column) gel filtration column. The antioxidant optimal component was filtered with a 0.45 μm microporous filter. 0.15 M sodium chloride was used as the eluent, and the elution flow rate was 0.5 mL / min. The ultraviolet detection wavelength was OD280 nm. Every 2 mL was collected in a tube. The number of tubes was combined according to the number of elution peaks, and the elution peaks were concentrated and freeze-dried.

[0068] For the high-activity component P3, reverse C18 chromatography column SunFire Prep C18 OBD TM(19 mm x 250 mm, 5 pm, Waters) column for further separation and purification. The preparative separation conditions were as follows: mobile phase A: 0.1% trifluoroacetic acid in water; mobile phase B: 0.1% trifluoroacetic acid in methanol; gradient elution program: gradient elution: 0-15 min, 30-40% B; 15-35 min, 40-90% B, injection volume: 500 pL; elution flow rate: 1 mL / min, column temperature: 30 °C, UV detection wavelength OD280 nm, and each fraction was collected and detected for DPPH and ABTS radical scavenging activity. + · Free radical scavenging activity. The component with the highest free radical scavenging rate after freeze-drying was collected, which was the Ganoderma lucidum spore powder antioxidant protein peptide, and its amino acid sequence was SEQ. ID. NO. 1-Phe-Ala-Asp-Glu-Asn-Phe-Lys.

[0069] The Ganoderma lucidum spore powder antioxidant protein peptide obtained in Examples 1-3 was subjected to the following detection / determination:

[0070] Nano-LC-MS / MS mass spectrometry detection:

[0071] The powder sample obtained in the above Examples 1-3 was injected into an EP tube, dissolved in 2 mL of mobile phase A (0.1% FA), vortexed to mix uniformly, and then ultrasonically treated in ice for 10 minutes. The mixture was centrifuged at 10000 g at 4 °C for 30 minutes, and the supernatant was collected and desalted by loading into a capture column Reprosil-Pur 120 C18 AQ (150 pm x 50 mm, 3 pm). Then, gradient elution was performed on an analytical column Reprosil-Pur 120 C18 AQ (150 pm x 170 mm, 1.9 pm) using 4-95% solvent B (0.1% FA, 80% ACN) for 66 min, with an injection volume of 1 pL and a flow rate of 600 nL / min. Tandem MS data were collected on a Thermo Fisher Q Exactive mass spectrometer equipped with a Nano Flex ion source using DDA (data-dependent acquisition) mass spectrometry technology. The mass spectrometry conditions were as follows: mass spectrometry primary electron energy 70 kV, full scan range m / z 100-15000, maximum injection time 100 ms. A maximum of 20 secondary mass spectra with charges +1 to +3 were collected per DDA cycle, with a mass spectrometry secondary electron energy of 17.5 kV, a maximum injection time of 50 ms, a precursor ion error of 20 ppm, and a daughter ion error of 0.02 Da. The search parameters were as follows: Ganoderma lucidum was selected in the Uniprot database; enzyme cleavage mode: nonspecific; and other parameters were default parameters.

[0072] The secondary mass spectrum of the polypeptide is as follows: Figure 3As shown, the parent ion is mainly fragmented into y and b ions, including y1 = 147.11, y2 = 294.18, y3 = 408.22, y4 = 537.27, y5 = 652.29, y6 = 723.33, b1 = 131.07, b2 = 219.11. The molecular weight of the first amino acid at the C-terminal of the peptide chain is y1-1 = 146, which is Lys (K); the molecular weight of the second amino acid from the C-terminal to the left is y2-y1+18 = 165, which is Phe (F); the molecular weight of the third amino acid is y3-y2+18 = 132, which is Asn (N); the molecular weight of the fourth amino acid is y4-y3+18 = 147, which is Glu (E); the molecular weight of the fifth amino acid is y5-y4+18 = 133, which is Asp (D); the molecular weight of the sixth amino acid is y6-y5+18 = 89, which is Ala (A); the molecular weight of the first amino acid from the N-terminal to the right is b1+17 = 148, which is Phe (F). In summary, the amino acid sequence of the active polypeptide is Phe-Ala-Asp-Glu-Asn-Phe-Lys.

[0073] Molecular docking simulation:

[0074] The three-dimensional crystal structure of the target protein Keap1 (PDB ID: 2FLU) was downloaded from the Protein Data Bank (PDB) database (http: / / www.rcsb.org), and the ligand file provided by the database was used as the standard. The receptor file was processed by removing water molecules and adding polar hydrogen using PyMOL software, and then the Nrf2 16-mer was removed, thereby obtaining an optimized receptor molecule. The small molecule polypeptide receptor was constructed using Chem draw 20.0 software, and hydrogen and charge were added. The MM2 molecular force field method was used to optimize the energy minimization to ensure that the polypeptide is close to the natural stable conformation. After the modeling of the ligand and receptor was completed, the pdb format file was saved for future use. The anti-oxidative peptide receptor sequence and Keap1 were subjected to semi-flexible molecular docking calculation by using the molecular docking software Discovery Studio 2019. The conformation with the lowest docking energy was selected as the best docking result, and the interaction force was analyzed by Discoveystudio and PyMOL. The secondary mass spectrum of the anti-oxidative peptide sequence and its molecular docking 2D results with Keap1 are shown in Figures 1 and 2, respectively. Figure 4 .

[0075] The Keap1 protein is the main negative regulator of cell protective gene expression, and the Nrf2 protein is the main positive regulator of cell protective gene expression. Activation of this pathway can activate the expression of a series of antioxidant enzymes including SOD, CAT, POD, and MDA, resulting in a decrease in ROS levels and cell protective effects in tissues. The binding energy (CDOCKER_ENERGY) of the positive control GSH and FADENFK with the Keap1 protein was -58.895 Kcal / mol and -134.11 Kcal / mol, respectively, indicating that FADENFK has a higher affinity for the Keap1 target and forms a strong interaction. Residues Arg326, Val561, and Cys368 have been reported as active sites of Keap1, and the sequence FADENFK can interact with two active site amino acids Arg326 and Cys368, forming a hydrogen bond with Arg326 and a hydrophobic force with Cys368. In addition, the Ganoderma lucidum spore polypeptide also forms hydrogen bonds with amino acid residues Gly367 and Val369, and a hydrophobic force with Val608 to maintain the stability of the complex. Therefore, the antioxidant pathway of the active peptide may be due to occupying the Nrf2 binding site on Keap1 to disrupt the Keap1-Nrf2 interaction, thereby activating the Nrf2 signaling pathway. In addition, in the docking results of FADENFK and GSH with Keap1, it was found that Ala556 participated in the formation of a hydrophobic force, and this amino acid may also be one of the key active amino acids of Keap1, which enhances the binding of FADENFK to Keap1.

[0076] Molecular dynamics simulation:

[0077] The two antioxidant peptide sequences with the lowest docking binding energy were subjected to molecular dynamics simulation using Amber 22 software. The topology of the polypeptide-protein (complex) was generated using the ff14SB force field, and to find the protein-polypeptide binding site, a TIP3P water-filled box was added, and Cl + or Na + ions were added to ensure that the simulation system was electrically neutral. The steepest descent method was used for energy minimization of the system to reduce unreasonable contacts or atomic overlaps in the entire system. After energy minimization, the simulation system was pre-equilibrated at 300K using the NVT module and the NPT module, and then the complex system was subjected to dynamic simulation for 100 ns. The trajectory after the simulation was completed was analyzed using the Pymol software, and the simulation trajectory was analyzed using the cpptraj program to analyze the trajectory parameters such as root mean square deviation (RMSD), root mean square fluctuation (RMSF), radius of gyration (Rg), and solvation surface area (SASA). The dynamics results of the antioxidant peptide complex are shown in Figures 5(a) to 5(e) .

[0078] From Figure 5(a) and Figure 5(b), it can be seen that within 20ns of the simulation initiation, the RMSD and RMSF show a rapid rising curve due to the conformation rearrangement caused by the binding of the polypeptide small molecule, and the complex system has not reached equilibrium, and converges into an equilibrium state at 20ns; both complexes fluctuate within the range of 0.7-1.2nm, and Keap1>FADENFK-Keap1, indicating that the RMSD of the polypeptide complex system is lower than that of the protein alone system, and the binding of the two stabilizes the protein conformation. Figure 5(c) shows that the Rg values of the two complex systems tend to be flat within the simulation time of 100ns, and with the addition of the active polypeptide small molecule, the protein Keap1 structure is caused to fold and become more compact, and with the extension of the simulation time, the small molecule continues to interact with the protein, and part of the domain unfolds to facilitate ligand binding, resulting in the unfolding of the protein domain and becoming loose, and the combination of the two promotes the binding of Keap1 to be more stable. Figure 5(d) shows that the SASA values of the two systems are generally stable within the simulation system of 100ns, and the average SASA value is Keap1>FADENFK-Keap1, indicating that the ligand FADEAKE is tightly wrapped by the binding pocket of the protein, with the least area exposed to the solvent, and the binding stability is higher. As can be seen from Figure 5(e), the active polypeptide contains a larger number of hydrogen bonds during the simulation process to maintain the stability of the system complex, which is also consistent with the molecular docking results. The results show that the stability of the heptapeptide complex FADENFK-Keap1 is better, which is also consistent with the molecular docking results.

[0079] Determination of reactive oxygen species:

[0080] The logarithmic phase HepG2 cells were digested with trypsin into a single cell suspension, and the cell concentration was adjusted to 5x10 4 The control group, the damage group and the low, medium and high dose groups of the antioxidant peptide were set in the 96-well plate, 200μL was inoculated in each well, and after 24h of culture in the incubator, the original culture medium was discarded, 200μL of complete culture medium containing different concentrations of polypeptide (12.5, 25, 50, 100μg / mL) was added to the experimental group, and only 200μL of complete culture medium was added to the damage group and the control group, and after 24h of culture in the CO2 incubator, they were washed twice with PBS, and 200μL and 400μmol / L of H2O2 were added to the experimental group and the damage group, respectively, and 200μL of complete culture medium was added to the control group, and after 30min of continuous culture, the cell survival rate was determined by CCK-8 method.

[0081] When the cell density reached 80%, the cells were divided into groups and treated with the drug for 24 hours. The next day, the fluorescent probe DCFH-DA (2', 7'-dichlorodihydrofluorescein bis ester) was diluted with serum-free medium at a ratio of 1:500 to achieve a final concentration of 10 μmol / L. The original culture medium was removed, and the diluted DCFH-DA solution was added. The cells were incubated at 37°C for 30 minutes. Then, the cells were washed three times with serum-free cell culture medium to completely remove the DCFH-DA that had not entered the cells. After washing, 200 μL of PBS was added to each well for subsequent detection. The fluorescence intensity was measured using a microplate reader with an excitation wavelength of 488 nm and an emission wavelength of 525 nm. The fluorescence distribution of the cells treated with different concentrations of polypeptides was observed under a fluorescence microscope.

[0082] The results of the fluorescence microscope are shown in Figure 6. Figures 6(a) to 6(g) The DCF fluorescence intensity of the drug administration group and the H2O2 model group was significantly higher than that of the control group. As the concentration of the polypeptide drug increased, the fluorescence intensity decreased significantly. This indicates that within the concentration range of 12.5-100 μg / mL, the polypeptide drug can significantly reduce the production of ROS in HepG2 cells. In addition, from the fluorescence microscope imaging results, it can be clearly seen that the morphology of the liver cancer cells changes from round to spindle-shaped, with small tentacles visible at the cell edge. As the concentration of the Ganoderma lucidum spore powder active polypeptide increases, the morphological changes become more obvious. This indicates that the polypeptide drug can cause significant morphological changes in HepG2 liver cancer cells, leading to apoptosis.

[0083] Determination of various antioxidant enzymes in cells:

[0084] The logarithmic phase HepG2 cells were digested with trypsin into a single cell suspension, and the cell concentration was adjusted to 5 × 10 4 The cells were incubated in a 12-well plate for 24 hours. After the cells were completely adherent, the original culture medium was discarded, and 2 mL of complete medium containing different concentrations of polypeptide (12.5, 50, 100 μg / mL) was added to the experimental group. The damage group and the blank group were added with 2 mL of complete medium. After 24 hours of culture, the cells were washed twice with PBS, and 2 mL and 400 μmol / L of H2O2 were added to the experimental group and the damage group, respectively. The blank group was added with 2 mL of complete medium. After 12 hours of continuous culture, the culture medium was discarded, and 500 μL of cell lysis solution was added to each well. The cells were centrifuged at 800 rpm for 5 minutes at 4°C, and the supernatant was collected and stored at -80°C. The protein content was determined using a BCA protein concentration determination kit, and the GSH, GSSG, MDA content and GSH-PX, SOD, CAT activity in the cell lysate were determined according to the instructions of the kit.

[0085] Table 1 Effects on antioxidant enzyme activity and MDA content in HepG2 cells

[0086]

[0087] Note: compared with the control group, *p<0.05, **p<0.01; compared with the model group, #p<0.05, ##p<0.01.

[0088] From Table 1, it can be seen that the MDA content in the H2O2-treated model group cells was significantly higher than that in the control group (p<0.05), while compared with the model group, the Ganoderma lucidum spore powder active peptide could significantly reduce the generation of MDA in the cells (p<0.05) in a concentration-dependent manner, indicating that the prepared active polypeptide could significantly reduce the MDA content of the damaged cells. Further, compared with the control group, the SOD, GSH-Px and CAT activities in the model group cells decreased by 61.81%, 55.76% and 76.85%, respectively. Compared with the model group, the addition of 12.5-100 μg / mL of Ganoderma lucidum spore powder active polypeptide into the HepG2 cells and incubation for a period of time, the SOD activity of the liver cancer cells was increased by 33.23%, 132.4% and 257.5%, respectively, the GSH-Px was increased by 11.35%, 69.55% and 97.07%, respectively, and the CAT activity was increased by 30.77%, 129.56% and 237.46%, respectively, indicating that the Ganoderma lucidum broken spore powder antioxidant protein peptide prepared by the present application could significantly increase the activity of antioxidant enzymes.

[0089] According to the above cell culture method, the prevention mechanism of H2O2-induced oxidative damage of HepG2 cells was studied, and the HepG2 cells were divided into a control group (D group), a model group (S group) and a polypeptide sample group (Y group), and the HepG2 cell culture method was the same as above. The polypeptide sample group: 100 μmol / L of Ganoderma lucidum broken spore powder antioxidant protein peptide was used to pre-incubate the cells for 4 h, and then the cells were incubated with a culture medium containing H2O2 (400 μmol / L) for 6 h, and then fresh culture medium was used for further culture for 6 h; the model group: the cells were incubated with a culture medium containing H2O2 (400 μmol / L) for 6 h, and then fresh culture medium was used for further culture for 6 h; the control group: normal HepG2 cells without Ganoderma lucidum broken spore powder antioxidant protein peptide and H2O2 were cultured for 12 h.

[0090] RIPA lysis buffer and phenylmethylsulfonyl fluoride (99:1, v / v) were added to each group of cells, vortexed, and then lysed on ice for 25 min. The supernatant was centrifuged at 13000 g at 4°C for 15 min, and the total protein content in the supernatant was determined using a BCA protein kit. After the same concentration of different groups of samples were separated by SDS-PAGE gel, the protein bands were transferred to PVDF membrane, blocked with 5% skim milk for 2 h, and then incubated with specific primary antibodies Nrf2 (1:1000 v / v), HO-1 (1:1000 v / v), and Keap1 (1:1000 v / v) at 4°C for 12 h. Then, the primary antibody was removed with TBST solution, washed 3 times, and then the PVDF membrane was incubated with secondary antibody containing horseradish peroxide (1:5000 v / v) for 1 h. After washing 3 times with TBST solution, ECL detection reagent was used for immunodetection, and the Quantity One software was used for protein quantification.

[0091] As shown in Figure 7(a), compared with the control group, the expression of Keap1 protein in the damaged HepG2 cells was significantly up-regulated (p<0.05), while the expression levels of Nrf2 and HO-1 proteins remained basically unchanged. Compared with the damaged cells, after treatment with different concentrations of ganoderma spore powder antioxidant peptides, the expression of Keap1 was significantly down-regulated (p<0.05), while the expression of Nrf2 and HO-1 proteins was significantly up-regulated (p<0.05), and the effect was further revealed in Figure 7(b) to be dose-dependent. This polypeptide can interact with the active residues of the Kelch domain of Keap1 through hydrogen bonds and hydrophobic interactions, thereby occupying the binding site of Nrf2 and Keap1, preventing the degradation of Nrf2 by protease, helping the nuclear transcription factor Nrf2 to enter the nucleus, and then activating the expression of downstream antioxidant gene HO-1, thereby playing a good antioxidant role, which is consistent with the above molecular docking results.

[0092] As shown in Table 2, compared with the control group, H2O2 treatment significantly reduced the GSH content in HepG2 cells (p<0.01) and increased the GSSG content (p<0.01). When further compared with the model group, after adding 12.5-100 μg / mL of Ganoderma lucidum spore powder antioxidant peptide into HepG2 cells and incubating for a period of time, the GSSG level of liver cancer cells was reduced by 52.12%-92.41%, and the GSH level was increased by 296.09%-737.24%. When the concentration of Ganoderma lucidum spore powder active peptide was 100 μg / mL, the GSH content was 36.42±2.03 nmol / mg prot, which was almost the same as the control group 37.44±1.26 nmol / mg prot, and the GSSG content was 1.16±0.05 nmol / mg prot, which was equivalent to the control group. The GSH / GSSG ratio in the last column of the table reveals that after adding Ganoderma lucidum spore powder active peptide, the ratio shows a rising trend and is concentration-dependent. Further, it is proved that after the H2O2-induced HepG2 cells are treated with Ganoderma lucidum spore powder antioxidant peptide, the oxidative damage can be alleviated by increasing GSH and inhibiting GSSG.

[0093] Table 2 Influence on GSSG, GSH content and their ratio of HepG2 cells

[0094]

[0095] Note: compared with the control group, *p<0.05, **p<0.01; compared with the model group, #p<0.05, ##p<0.01.

[0096] The present application extracts Ganoderma lucidum spore protein from Ganoderma lucidum spore powder residue as raw material, obtains crude peptide hydrolysate by trypsin, obtains antioxidant peptide sequence through separation, purification and peptide spectrum identification, and tests the antioxidant activity through H2O2-induced HepG2 cell oxidative damage model. The Ganoderma lucidum spore powder antioxidant peptide can activate the Keap1-Nrf2 oxidative stress signal pathway of liver cancer cells, significantly improve the intracellular oxidase activity (SOD, GSH-Px, CAT) and GSH / GSSG ratio, inhibit the generation of ROS free radicals, reduce the cell membrane damage and MDA content, and thus effectively play the antioxidant function. The present application provides a new idea for the development and utilization of protein and polypeptide resources of Ganoderma lucidum spore powder, and has a good application prospect in the research and development of functional food, medicine and health products.

[0097] Although the present application has been disclosed as above, it is not intended to limit the protection scope of the present application, and any modification and improvement made by those skilled in the art without departing from the concept and scope of the present application shall belong to the protection scope of the present application.

Claims

1. A type of antioxidant protein peptide from Ganoderma lucidum spore powder with broken cell wall, characterized in that, Its amino acid sequence is SEQ.ID.NO.1-Phe-Ala-Asp-Glu-Asn-Phe-Lys.

2. A method for preparing antioxidant protein peptides from Ganoderma lucidum spore powder with broken cell walls, characterized in that the steps are as follows: as follows: (1) Raw material pretreatment: The residue after processing Ganoderma lucidum spore powder is used as raw material. Chitinase with an enzyme amount of 2000-4000 U / g is added to the raw material at a material-to-liquid ratio of 1:20 (g / mL). The raw material is treated for 2-3 hours under the extraction conditions of 45℃ and pH value adjusted to 6.0-8.

0. After the reaction is completed, the enzyme is inactivated by boiling water bath for 10 minutes. The raw material is filtered while hot and the filter residue is retained. (2) Protein extraction: The filter residue and deionized water were mixed at a ratio of 1:10-1:20 (g / mL). The pH was adjusted to 8.5-10.5 with 1 mol / L NaOH. The extraction time was 2-4 h and the temperature was 40-60℃. After centrifugation at 8000 r / min for 15 min, the supernatant was collected. The supernatant was concentrated to 1 / 3 of its volume by rotary evaporation at 50℃. H2O2 (V / V) with a volume fraction of 6% was added for decolorization for 3 h. The pH was adjusted to the isoelectric point of 2.5-4 with 0.1 mol / L HCl solution. The precipitated protein was collected, washed with deionized water several times until neutral, and then freeze-dried. (3) Biological enzymatic hydrolysis: Trypsin was used. The protein and deionized water were mixed at a ratio of 1:10-1:30 (g / mL). The enzyme addition was 6000-9000 U / g. The hydrolysis time was 2-4 h, the hydrolysis temperature was 40-60℃, and the pH value was 7.5-8.

5. After the hydrolysis reaction was completed, the enzyme was inactivated at 100℃ for 10 min to stop the reaction. After cooling to room temperature, the mixture was centrifuged at 8000 r / min for 15 min. The supernatant was collected and the pH value was adjusted to 7.0 to obtain the hydrolysate for later use. (4) Ultrafiltration membrane separation: Use an ultrafiltration membrane with a molecular weight cutoff of 3KD to separate the enzymatic hydrolysate and collect crude peptide enzymatic hydrolysate with a molecular weight ≤3kDa; (5) Continuous chromatographic separation: The crude peptide hydrolysate was separated by dextran gel chromatography column elution; then further purified by reversed C18 chromatography column, with each 1 mL tube collected after freeze-drying. + The component with the highest free radical scavenging rate was used to prepare the antioxidant protein peptide from Ganoderma lucidum spore powder with the amino acid sequence SEQ.ID.NO.1-Phe-Ala-Asp-Glu-Asn-Phe-Lys.

3. The preparation method according to claim 2, characterized in that, The parameters for ultrafiltration membrane separation and purification in step (4) are: pressure difference of 0.1-0.5 MPa, degradation solution temperature of 15-50℃, and membrane area of ​​1.5-5 m². 2 The membrane flux of the enzymatic hydrolysate is 0.1-2.5 m. 2 / h; Ultrafiltration uses tangential flow for membrane separation.

4. The preparation method according to claim 3, characterized in that, The parameters for ultrafiltration membrane separation and purification in step (4) are: pressure difference of 0.15-0.3 MPa, degradation solution temperature of 20-30℃, and membrane area of ​​1.5-2.5 m². 2 The membrane flux of the enzymatic hydrolysate is 0.2-0.8 m. 2 / h.

5. The preparation method according to claim 2, characterized in that, The dextran gel in step (5) was prepared using a Sephadex G-25 gel chromatography column (XK 16 / 70mm, Pharmacia), with an elution flow rate of 0.5 mL / min and an ultraviolet detection wavelength of OD280 nm. Each tube contained 2 mL of gel.

6. The preparation method according to claim 2, characterized in that, The reverse C18 column used in step (5) was a SunFirePrep C18 OBD. TM (19mm×250mm×5μm, Waters) chromatographic column, prepared under the following separation conditions: mobile phase A: 0.1% aqueous solution of trifluoroacetic acid; mobile phase B: 0.1% methanol solution of trifluoroacetic acid; gradient elution program: gradient elution: 0–15 min, 30–40% B; 15–35 min, 40–90% B; injection volume: 500 μL; elution flow rate: 1 mL / min; column temperature: 30℃; each fraction was detected and collected at a detection wavelength of 280 nm.

Images