Blood-glucose-reducing and anti-oxidation black fungus protein as well as preparation method and application thereof

Abstract

The technical scheme of the invention discloses black fungus protein capable of reducing blood glucose and resisting oxidation as well as a preparation method and application of the black fungus protein. The amino acid sequence of the black fungus protein is as shown in SEQ ID NO. 1. The protein which is separated and identified from the black fungus for the first time and has the effects of reducing blood glucose and resisting oxidation achieves the effect of reducing blood glucose through double inhibition of alpha-amylase and alpha-glucosidase, meanwhile, the protein has remarkable antioxidant activity, animal experiments prove that fasting blood glucose and body weight of a type II diabetes model mouse can be effectively reduced, and the protein has a good application prospect. The compound has high biological activity and safety, and can be used for developing functional products with hypoglycemic and antioxidant activity.

Description

TECHNICAL FIELD

[0001] The present application relates to the technical field of biological medicine and functional food, and particularly relates to a black fungus protein with hypoglycemic and antioxidant effects and a preparation method and application thereof. BACKGROUND

[0002] As a traditional fungus with both medicinal and edible functions, black fungus is rich in active ingredients such as polysaccharides, proteins, flavonoids and dietary fiber. The polysaccharide component of black fungus has been widely studied and applied in the fields of metabolic syndrome intervention and immune regulation. The protein content in black fungus accounts for 15-20% of the dry weight, and the protein has significant advantages in biological activity and absorption efficiency. However, the functional research on the protein in black fungus has been less for a long time. At present, no black fungus protein with clear amino acid sequence and both hypoglycemic and antioxidant effects has been successfully separated and identified.

[0003] Patent CN111217893 A discloses a black fungus agglutinin and a preparation method thereof. A new protein, black fungus agglutinin LAA, in black fungus is obtained by preparing a black fungus agglutinin crude extract, mixing the black fungus agglutinin crude extract with pig stomach mucin type II-cyanogen bromide activated agarose gel, centrifuging and dissociating, ultrafiltrating and freeze-drying. The black fungus agglutinin LAA is identified by protein identification and compared with the UniProt database. It is analyzed that the black fungus agglutinin LAA contains the sequences of 4 peptide segments. The black fungus agglutinin can inhibit the growth of cancer cells, promote the release of NO, IL-1β and TNF-α of macrophages, has the activity of improving immunity, can remove ABTS free radicals, hydroxyl free radicals and superoxide anions, and has excellent antioxidant effect. The research has made a certain progress. However, further exploration of the stereostructure, mechanism and function of the protein requires more detailed protein information, especially the protein sequence. At present, no black fungus protein with complete amino acid sequence and both hypoglycemic and antioxidant effects has been reported. Therefore, it is necessary to separate and identify the black fungus protein, determine the amino acid sequence thereof, study the function thereof and develop and apply the black fungus protein. SUMMARY

[0004] In order to solve the existing technical problems, the present application provides a black fungus protein with hypoglycemic and antioxidant effects and a preparation method and application thereof. The protein is separated and identified from black fungus, the sequence of the black fungus protein is obtained, and it is verified that the black fungus protein has significant hypoglycemic and antioxidant effects.

[0005] In order to achieve the above application purposes, the present application provides the following technical solutions.

[0006] The present application provides a black fungus protein with hypoglycemic and antioxidant effects. The amino acid sequence of the black fungus protein is shown in SEQ ID NO. 1.

[0007] This invention also provides a method for preparing black fungus protein that has hypoglycemic and antioxidant properties, comprising the following steps:

[0008] S1. Pre-treatment of black fungus: Dry the black fungus, grind it into black fungus powder, sieve it, and store it for later use.

[0009] Furthermore, in S1, the black fungus is dried black fungus, which is dried in an electric heating drying oven and then pulverized into black fungus powder using a functional pulverizer.

[0010] Preferably, the drying temperature in step S1 is 55°C, the drying time is 8 hours, and the sieve is a 100-mesh sieve.

[0011] S2. Extraction of black fungus protein: Add black fungus powder to NaOH solution, stir evenly, extract by ultrasonication in a 40℃ water bath, collect the supernatant by centrifugation, and obtain black fungus protein extract.

[0012] Preferably, in step S2, 5g of black fungus powder is weighed and added to 0.007 M NaOH solution at a material-to-liquid ratio of 1:90 (m / V), and stirred evenly; ultrasonic extraction is performed at 100 w for 1.5 h; centrifugation is performed at 8000 r / min for 20 min, and the supernatant is collected.

[0013] S3. Purification of black fungus protein: The black fungus protein extract was filtered to remove impurities, subjected to ion exchange column chromatography, and each elution peak was collected separately. Each elution peak was freeze-dried, and the α-glucosidase inhibition rate and α-amylase inhibition rate were determined. The fraction with the highest α-glucosidase inhibition rate and α-amylase inhibition rate was selected to obtain the target purified fraction.

[0014] Furthermore, in S3, the ion exchange column chromatography is performed using a Q-Sepharose Fast Flow anion exchange column with a column volume of 80 mL.

[0015] Preferably, filter paper is used for filtration in step S3.

[0016] Preferably, the chromatography in step S3 includes the following steps: first, equilibrate the column with equilibration buffer, then load the sample after equilibration, and then wash away unbound impurities on the column with equilibration buffer; perform linear gradient elution with 3-5 column bed volumes of eluent at 0-100%, the equilibration buffer being 20 mM Na2HPO4-NaH2PO4 buffer with a pH of 5.8; the eluent being 1M NaCl + 20 mM Na2HPO4-NaH2PO4 buffer with a pH of 5.8; the equilibration and sample loading flow rate being 1.8 ml / min, and the elution flow rate being 3.0 ml / min; the detection wavelength being 280 nm, and the chromatographic curve being recorded.

[0017] S4. Secondary purification of black fungus protein: The target purified component prepared in S3 was dissolved in deionized water to prepare a target purified component solution, and purified by gel filtration chromatography. According to the elution curve results, different elution components were obtained and electrophoresed separately. The elution components with target protein bands were dialyzed and lyophilized to obtain purified black fungus protein.

[0018] Furthermore, the purification process in S4, which was performed by gel filtration chromatography, is now performed by a Sephacryl S-100 gel chromatography column.

[0019] Preferably, in step S4, 200 mg of the target purified component prepared in step S3 is weighed and dissolved in 2 ml of deionized water to prepare a 100 mg / ml target purified component solution.

[0020] Preferably, the purification of the Sephacryl S-100 gel chromatography column in S4 includes the following steps: filtering and rinsing the Sephacryl S-100 gel medium to remove fragments and impurities, then packing the gel into a glass column, settling and compacting it, eluting the gel chromatography column with 3-5 column volumes of deionized water, equilibrating the gel chromatography column with PBS buffer, loading the sample after equilibration, eluting the gel chromatography column with PBS buffer, and recording the elution curve; the PBS buffer is 50 mM NaCl-20 mM Na2HPO4-NaH2PO4 buffer, pH 6.8, the elution flow rate is 0.6 ml / min, and the detection wavelength is 280 nm.

[0021] Preferably, the glass column in S4 is 1.6 × 100 cm.

[0022] Application of a hypoglycemic and antioxidant black fungus protein in the preparation of functional products with hypoglycemic and antioxidant activities.

[0023] By adopting the above technical solution, the beneficial effects achieved by the present invention are as follows:

[0024] (1) The amino acid sequence of the black fungus protein of the present invention has not been reported. The molecular weight is 23.558 kDa. The comparison with the NCBI database confirms that the homology with known proteins is ≤87.72%. It is the first protein isolated and identified from black fungus that has hypoglycemic effect, antioxidant activity and weight reduction.

[0025] (2) The black fungus protein of the present invention achieves hypoglycemic effect by dual inhibition of α-amylase and α-glucosidase, and at the same time has significant antioxidant activity. Animal experiments have confirmed that it can effectively reduce fasting blood glucose and weight in type II diabetic model mice. It can be used to develop hypoglycemic drugs, health products or functional oral products, and provide new methods and applications for the prevention and treatment of diabetes and obesity. Attached Figure Description

[0026] Figure 1 The elution curve of the black fungus protein powder in Example 1 using Q-Sepharose Fast Flow anion exchange chromatography column is shown.

[0027] Figure 2 The elution curve of the target purified component in Example 1 using Sephacryl S-100 gel chromatography column;

[0028] Figure 3 Electrophoresis diagrams of peaks S1, S2, S3, and S4 from Example 1;

[0029] Figure 4 The image shows the HPLC chromatogram of purified black fungus protein from Example 1.

[0030] Figure 5 This is a mass spectrum of the purified black fungus protein from Example 1. Detailed Implementation

[0031] The present invention will be further described in detail below with reference to specific embodiments.

[0032] Example 1

[0033] A method for preparing black fungus protein with hypoglycemic and antioxidant properties includes the following steps:

[0034] S1. Pre-treatment of black fungus: After drying the dried black fungus in an electric heating drying oven, remove it and cool it. Use a multi-functional grinder to grind the black fungus into black fungus powder, then sieve the black fungus powder and store it for later use.

[0035] Preferably, the drying temperature in step S1 is 55°C, the drying time is 8 hours, and the sieving is done through a 100-mesh sieve.

[0036] S2. Extraction of black fungus protein: Mix black fungus powder with NaOH solution evenly, extract by ultrasonication in a 40℃ water bath, and then centrifuge to collect the supernatant to obtain black fungus protein extract.

[0037] Preferably, in step S2, 5 g of black fungus powder and 0.007 M NaOH solution are mixed evenly at a material-to-liquid ratio of 1:90 (m / V), ultrasonicated at 100 W for 1.5 h, centrifuged at 8000 r / min for 20 min.

[0038] S3. Purification of black fungus protein: The black fungus protein extract was filtered through filter paper to remove impurities. The filtered black fungus protein solution was then subjected to chromatographic separation using a Q-Sepharose Fast Flow anion exchange column (column volume: 80 ml). The elution peaks were collected in the order of elution. The elution peaks were lyophilized separately, and the α-glucosidase inhibition rate and α-amylase inhibition rate were determined. The fraction with the highest α-glucosidase inhibition rate and α-amylase inhibition rate was selected as the target purified fraction.

[0039] Preferably, in step S3, filter paper is used for filtration, and the column volume of the Q-Sepharose Fast Flow anion exchange chromatography column is 80 ml.

[0040] Preferably, the chromatography in step S3 includes the following steps: first, equilibrate the column with equilibration buffer, then load the sample after equilibration, and then wash away unbound impurities on the column with equilibration buffer; perform linear gradient elution with 3-5 column bed volumes of eluent at 0-100%, the equilibration buffer is 20 mM Na2HPO4-NaH2PO4 buffer with a pH of 5.8, and the eluent is 1 M NaCl + 20 mM Na2HPO4-NaH2PO4 buffer with a pH of 5.8; the equilibration and sample loading flow rate is 1.8 ml / min, and the elution flow rate is 3.0 ml / min; the detection wavelength is 280 nm, and the chromatographic curve is recorded.

[0041] Preferably, the elution peaks in S3 are the elution peaks of three protein components, labeled as Q1, Q2 and Q3, respectively, among which Q1 has the highest α-glucosidase inhibition rate and α-amylase inhibition rate.

[0042] S4. Secondary separation and purification of black fungus protein: The target purified component prepared in S3 was dissolved in deionized water to prepare a target purified component solution, and purified by Sephacryl S-100 gel chromatography column; according to the elution curve results, different elution components were obtained, and each elution component was subjected to electrophoresis. The elution components with protein bands were dialyzed and lyophilized to obtain purified black fungus protein.

[0043] Preferably, in step S4, 200 mg of the target purified component prepared in step S3 is weighed and dissolved in 2 ml of deionized water to prepare a 100 mg / ml target purified component solution.

[0044] Preferably, the purification of the Sephacryl S-100 gel chromatography column in S4 includes the following steps: filtering and rinsing the Sephacryl S-100 gel to remove fragments and impurities, then packing the gel into a glass column and compacting it to form a gel chromatography column to prevent the generation of bubbles or fragments, then eluting the gel chromatography column with deionized water for 3-5 column volumes, equilibrating the gel chromatography column with PBS buffer, loading the sample after equilibration, eluting the gel chromatography column with PBS buffer, and recording the elution curve; the PBS buffer is 50 mM NaCl-20 mM Na2HPO4-NaH2PO4 buffer with a pH of 6.8, an elution flow rate of 0.6 ml / min, and a detection wavelength of 280 nm.

[0045] Preferably, the glass column in S4 is 1.6 × 100 cm.

[0046] Preferably, four elution components are obtained in S4, namely peak S1, peak S2, peak S3 and peak S4.

[0047] Preferably, the elution fractions S1, S2, S3, and S4 in S4 are subjected to sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE), comprising the following steps: loading 16 μL of the sample solution to be tested into each well, with a sample solution concentration of 20 mg / ml; performing electrophoresis in NuPAGE MES-SDS electrophoresis buffer; selecting a 12% separating gel and a 6% stacking gel; staining with 1% Coomassie Brilliant Blue R250 for 1 h; and then treating with a destaining solution containing 10% acetic acid for 40 min. The electrophoresis results are as follows: Figure 3 As shown, from Figure 3 The results show that among the components S1, S2, S3 and S4, only the S2 component has the corresponding protein band, indicating that among the components eluted by Sephacryl S-100 gel chromatography, only the S2 peak is the purified black fungus protein.

[0048] 1. Determination of the sequence, molecular weight, and purity of proteins from black fungus.

[0049] 1.1 High Performance Liquid Chromatography Analysis

[0050] 20 mg of the purified black fungus protein prepared in step S4 was dissolved in 1 ml of PBS buffer to prepare a 20 mg / ml sample solution. The purity of this solution was determined by high-performance liquid chromatography (HPLC). The chromatographic column was a PROTEIN KW-802.5 column (8*300 mm), the sample loading volume was 10 μL, and the mobile phase was PBS buffer (150 mM NaCl-20 mM Na2HPO4-NaH2PO4 buffer, pH 7.0). The elution flow rate was 1 ml / min, the detection wavelength was 215 nm, and isocratic elution was performed for 20 min. The chromatogram was recorded, and the results are shown below. Figure 4 As shown.

[0051] from Figure 4 The results show that the purified black fungus protein prepared in step S4 was identified as a single component with a unique retention value by high performance liquid chromatography. The retention time of the purified black fungus protein was 8 min, and the purity was 100% according to the normalization method. That is, the purity of the purified black fungus protein prepared in step S4 was 100%.

[0052] 1.2. Molecular weight determination by mass spectrometry (LC-MS) (commissioned to Shanghai Microspectrometry Testing Technology Group Co., Ltd.)

[0053] The purified black fungus protein prepared in step S4 was detected using a Thermo Vanquish-Q Exactive Plus ultra-high performance liquid chromatography-mass spectrometry system, and analyzed using Biopharma Finder software. The molecular weight of the purified black fungus protein was determined to be 23.558 kDa.

[0054] One purified black fungus protein sample was taken, 1 ml of purified water was added, and after mixing, 100 µl was taken into an intratubation tube for analysis. The injection volume was 40 μl. The sample was separated using an ultra-high performance liquid chromatography (UHPLC) system. Mobile phase A was 0.1% FA aqueous solution, and mobile phase B was 0.1% FA acetonitrile solution. The column was equilibrated with the initial gradient of mobile phase A. The sample was loaded by an autosampler and then separated by the column gradient at a flow rate of 0.3 ml / min, a detection wavelength of 280 nm, and a column temperature of 80℃. The chromatographic column used was an ACQUITYUPLC Protein BEHC4 column, 300 Å, 1.7 µm, 2.1 mm * 50 mm. The relevant liquid phase gradient was 0-11 min corresponding to 10% B-90% B, and 11.1-15 min corresponding to 10% B. The test sample was analyzed by mass spectrometry using a Thermo Vanquish-Q Exactive Plus ultra-high performance liquid chromatography-mass spectrometry system. The sample was processed using Biopharm Finder software with the method type set to Intact Mass Analysis. The purified black fungus protein sample prepared in step S4 was analyzed by LC-MS and the measured molecular weight was 23558.56250 Da. The results are as follows: Figure 5 As shown.

[0055] 1.3 Protein N-terminal sequencing: (commissioned to Shanghai MicroSpectrum Detection Technology Group Co., Ltd.)

[0056] The first 15 amino acids of the N-terminal sequence of the purified black fungus protein prepared in step S4 were analyzed using a Shimadzu PPSQ-53A fully automated protein and peptide sequencer. The N-terminal amino acid sequence was compared with the theoretical N-terminal amino acid sequence to determine its accuracy. The specific experimental steps were as follows: 1 ml of purified water was added to the sample vial of the purified black fungus protein, and the mixture was vortexed. Then, 300 μl of the sample was transferred to a 3 kD ultrafiltration tube and ultrafiltered to a final volume of approximately 100 μl. Finally, 30 μl of the concentrated sample was transferred onto a PVDF membrane for analysis. The N-terminal sequence of the first 15 amino acids of the purified black fungus protein sample prepared in step S4 was determined using the Edman method. The obtained amino acid sequence was RNIVVKNNPPFTIWP.

[0057] 1.4. Denovo sequencing (contracted to third-party Baizhen Biotechnology (Wuhan) Co., Ltd.)

[0058] A suitable amount of purified black fungus protein prepared using S4 was subjected to in-solution enzymatic hydrolysis. First, TCEP was added to the sample to reduce the protein, followed by alkylation with iodoacetamide. Then, the sample was digested with multiple enzymes (trypsin, chymotrypsin, and pepsin). After the enzymatic hydrolysis reaction, the sample was desalted using a C18 desalting column, lyophilized, reconstituted with 0.1% formic acid solution, and then analyzed by mass spectrometry. The mass spectrometry analysis procedure was as follows: after TCEP reduction and glycolysis by PNGase F enzyme, the sample was analyzed by LC-MS using a Thermo Scientific Orbitrap Fusion Tribrid high-resolution mass spectrometer equipped with a positive ion mode heated electrospray ionization source and a Thermo Fisher Ultimate 3000 RSLCnano HPLC system. The enzymatic hydrolysis products were analyzed on an Orbitrap mass spectrometer (Orbitrap Fusion or Eclipse, Thermo Fisher Scientific). Both full MS and MS2 scans were acquired on a high-resolution Orbitrap mass spectrometer. Fragment ions were generated using HCD and EThcD fragmentation modes to obtain MS2 data. All raw data files acquired by mass spectrometry were analyzed using PEAKS AB 3.5. The final amino acid sequence of the purified black fungus protein was determined to be: RNIVVKNNCPFTIWPALFTTPGGPIPDAPTGWEAASGSSQTITVPENWTSGRIWGRTGCDFSTSLPGPTQCETGGCNGGLLCDPTTGTGVPPASLAEWTLGDQDSYDVSLVDGSNLPIRVEAANCPTGECPVDLNPNCPAELQAKNAAGAIVGCKSACFANLDGNPGDSANCCSGSHNTPQTCPSSGVQDYDYFHSACPNAYAYAYDDTTALKFCSAHSDYTLTFCP.

[0059] The amino acid sequence of black fungus protein was compared with that in the NCBI database. The comparison revealed that there was no protein sequence in the NCBI database that completely overlapped with the purified black fungus protein, with the highest homology being 87.72%.

[0060] 2. Functional verification of black fungus protein

[0061] 2.1 Determination of α-glucosidase inhibition activity

[0062] Add 50 μL of 2U α-glucosidase solution and 50 μL of sample solution sequentially to a 96-well plate. Incubate the mixture at 37°C for 20 min. Add 50 μL of 5 mM PNPG (p-nitrophenyl glucosyl pyranoside) solution and continue incubation at 37°C for 30 min. Then, add 50 μL of 1% CaCO3 solution to each well to stop the reaction. Measure the absorbance of each well at 405 nm using a microplate reader and calculate the α-glucosidase activity inhibition rate (%) of the sample according to formula (1).

[0063] Formula (1) is: α-glucosidase activity inhibition rate (%) = [(AB) - (CD)] / (AB) × 100

[0064] Note: In formula (1), group (A) uses PBS buffer instead of the test sample solution, blank control group (B) uses PBS buffer instead of the enzyme solution and the test sample solution, and background control group (D) uses PBS buffer instead of the enzyme solution.

[0065] The sample group consisted of three batches of samples for which the inhibition rate of α-glucosidase activity needed to be determined. These included the black fungus protein extract (concentration of 20 mg / ml) prepared in step S2 of Example 1, and components Q1, Q2, and Q3 (concentration of 50 mg / ml) eluted by Q-Sepharose Fast Flow anion exchange chromatography in step S3 of Example 1. Samples Q1, Q2, and Q3 contained salt, while the black fungus protein extract was salt-free. Therefore, the concentration of the samples was limited, and acarbose with a concentration of 10 ng / ml was used as the control group. The inhibition rate of α-glucosidase activity of the three batches of samples was calculated according to formula (1), and the results are shown in Table 1.

[0066] Table 1. Results of α-glucosidase activity inhibition in each component

[0067] Group Inhibition rate Acarbose 50.75%±4.41% Black fungus protein extract 22.47%±7.65% Q1 38.37%±9.11% Q2 1.51%±2.68% Q3 0.14%±9.05%

[0068] As can be seen from the results in Table 1, among the black fungus protein extract prepared in step S2 of Example 1 and components Q1, Q2 and Q3 in step S3 of Example 1, component Q1 showed the highest inhibition rate against α-glucosidase activity.

[0069] 2.2 Determination of α-amylase inhibition activity

[0070] Add 0.1 ml of α-amylase solution (concentration of 40 U / ml) and 0.1 ml of sample solution to a 2 ml centrifuge tube in sequence. Incubate the mixture at 37°C for 20 min. Add 0.1 ml of starch solution with a mass percentage concentration of 1% and continue to incubate the mixture at 37°C for 40 min. Then add 50 μl of DNS solution to each tube and boil at 100°C for 10 min to stop the reaction. After the liquid in the centrifuge tube returns to room temperature, aspirate 150 μL of liquid from each tube and place it in a 96-well plate. Measure the absorbance of each well at 540 nm using a microplate reader and calculate the α-amylase activity inhibition rate (%) of the sample according to formula (2).

[0071] Formula (2) is: α-amylase activity inhibition rate (%) = [(AB) - (CD)] / (AB) × 100

[0072] Note: In formula (2), group (A) uses PBS buffer instead of the test sample solution, blank control group (B) uses PBS buffer instead of the enzyme solution and the test sample solution, and background control group (D) uses PBS buffer instead of the enzyme solution.

[0073] The sample group consisted of three batches of samples for which the α-amylase activity inhibition rate needed to be determined. These included the black fungus protein extract (concentration 20 mg / ml) prepared in step S2 of Example 1, components Q1, Q2, and Q3 (concentration 50 mg / ml) eluted by Q-Sepharose Fast Flow anion exchange chromatography in step S3 of Example 1, and acarbose at a concentration of 10 ng / ml as a control group. The α-glucosidase activity inhibition rate of the three batches of samples was calculated according to formula (2), and the results are shown in Table 2.

[0074] Table 2. Results of α-amylase activity inhibition in each component

[0075] Group Inhibition rate Acarbose 53.75%±20.58% Black fungus protein extract 34.71%±12.87% Q1 65.11%±5.75% Q2 6.17%±4.00% Q3 19.50%±7.27%

[0076] As shown in Table 2, among the black fungus protein extract prepared in step S2 of Example 1, and components Q1, Q2, and Q3 in step S3 of Example 1, component Q1 exhibited the highest inhibition rate against α-amylase activity. Combining the results in Tables 2 and 1, it is clear that component Q1 possesses the highest hypoglycemic activity. Therefore, component Q1 was selected for dialysis and freeze-drying to obtain the target purified component.

[0077] 2.3 Determination of the inhibitory activities of α-glucosidase and α-amylase on purified black fungus protein

[0078] The experimental conditions for inhibiting α-glucosidase activity and α-amylase activity were as described in 2.1 and 2.2. The sample group consisted of purified black fungus protein prepared in step S4 of Example 1, dissolved in deionized water to prepare a sample solution with a concentration of 50 mg / ml. Acarbose with a concentration of 10 ng / ml was used as the control group. The experimental results are shown in Table 3.

[0079] Table 3. Inhibition rates of α-glucosidase and α-amylase activities of purified black fungus protein.

[0080] Group Inhibition rate of α-glucosidase activity Inhibition rate of α-amylase activity Acarbose 49.17%±3.37% 49.50%±13.68% Purified black fungus protein 26.27%±14.01% 58.55%±6.91%

[0081] As can be seen from the results in Table 3, the purified black fungus protein has good inhibitory activity against α-glucosidase and α-amylase. Therefore, the purified black fungus protein prepared in step S4 of Example 1 has very good hypoglycemic activity.

[0082] 2.4 Determination of the antioxidant activity of purified black fungus protein

[0083] 2.4.1 Determination of DPPH free radical scavenging ability

[0084] Take 400 μL of the test solution, add 600 μL of DPPH • 80% methanol working solution, and let stand in the dark for 30 min. Measure the absorbance A1 (517 nm). Mix 400 μL of the test solution and 80% methanol solution, and measure the absorbance A2. Mix 400 μL of 80% methanol solution and 600 ml of DPPH • 80% methanol solution, and measure the absorbance A3. The DPPH free radical scavenging rate is calculated according to formula (3).

[0085] Formula (3) is: DPPH free radical scavenging rate (%) = (1 - (A1 - A2) ÷ A3) × 100%;

[0086] The test solution was prepared by dissolving the purified black fungus protein prepared in step S4 of Example 1 in deionized water to a concentration of 50 mg / ml. Vitamin C with a concentration of 100 ng / ml was used as a control group. The experimental results are shown in Table 4.

[0087] 2.4.2 Determination of ABTS free radical scavenging ability

[0088] Add 10 μL of the test solution to a 96-well plate, add 200 μL of ABTS working solution, mix well, react for 2-6 min, and measure the absorbance value A1 at 734 nm; use 80% ethanol instead of the working solution and distilled water instead of the test solution to measure the absorbance values ​​A2 and A3; calculate the ABTS free radical scavenging rate according to formula (4).

[0089] Formula (4) is: ABTS free radical scavenging rate (%) = (1 - (A1 - A2) ÷ A3) × 100%;

[0090] The test solution was prepared by dissolving the purified black fungus protein prepared in step S4 of Example 1 in deionized water to a concentration of 50 mg / ml. Vitamin C with a concentration of 100 ng / ml was used as a control group. The experimental results are shown in Table 4.

[0091] Table 4. DPPH and ABTS free radical scavenging rates of purified black fungus protein

[0092] Group DPPH radical scavenging rate ABTS radical scavenging rate Vc 84.88%±2.3% 86.50%±7.6% Purified black fungus protein 39.87%±3.0% 43.16%±7.0%

[0093] The results in Table 4 show that the purified black fungus protein has good DPPH and ABTS free radical scavenging rates, indicating that the purified black fungus protein prepared in step S4 of Example 1 has good antioxidant activity.

[0094] 2.5 Effects of purified black fungus protein on blood glucose and body weight in type II diabetic mice

[0095] Fifty 4-week-old C57BL / 6L mice (provided by Jiangsu Huachuang Xinno Pharmaceutical Technology Co., Ltd.) were selected and acclimatized for one week before being randomly divided into a model group and a normal group. Ten mice in the normal group were fed a standard diet, while the remaining mice in the model group were fed a high-fat, high-sugar diet. After 8 weeks of feeding, the mice were fasted for 12 hours (overnight fasting, but water was allowed), and then injected intraperitoneally with 55 mg / kg of STZ. Blood glucose levels were measured randomly for three consecutive days 72 hours after STZ injection. A blood glucose level >16.7 mmol / L (or a fasting blood glucose level ≥11.1 mmol / L for three consecutive days) was considered a successful diabetes mellitus (DM) model. The mice in the model group were then divided into four groups of 10 mice each based on their blood glucose levels.

[0096] The mice were divided into four groups: a black fungus powder group (1850 mg / kg body weight), a black fungus protein group (30 mg / kg body weight), a smegglutinin group (10 mg / kg body weight), a blank control group, and a normal control group. The black fungus powder group received the black fungus powder prepared in step S1 of Example 1; the black fungus protein group received the purified black fungus protein prepared in step S4 of Example 1; the smegglutinin group received smegglutinin; and the blank control group and normal control group received ordinary feed. The administration was by gavage, once daily for 5 weeks, with continuous monitoring of blood glucose and body weight. The body weight of mice in each group at weeks 0, 1, 2, 3, 4, and 5 after administration is shown in Table 5; the blood glucose results are shown in Table 6.

[0097] Table 5. Changes in body weight of mice in each group at different drug administration times.

[0098] ( ± SD, n=10, unit: g)

[0099] Group Blank control group Normal group Black fungus powder group Black fungus protein group Semaglutide group 0 weeks 28.4±1.3 23.7±1.5** 29.3±0.9 29.1±0.9 28.6±0.8 1 week 27.9±1.3 24.3±1.4** 28.7±0.8 28.4±1.0 28.0±0.8 2 weeks 28.7±1.3 24.8±1.3** 28.1±0.9 28.9±1.0 28.4±0.9 3 weeks 29.8±1.4 25.2±1.3** 28.5±0.9** 29.3±1.0 28.7±0.9 4 weeks 30.6±1.4 25.6±1.2** 28.8±0.9** 29.6±1.0 29.1±0.8** 5 weeks 31.6±1.3 26.0±1.3** 29.2±0.9** 30.0±1.0** 29.2±0.8**

[0100] Note: Compared with the model group, *P<0.05, **P<0.01

[0101] As shown in Table 5, the body weight of mice in the blank control group continued to increase over a 5-week period. After administration of the test substance, the body weight of diabetic model mice in the fungus powder group, fungus protein group, and smegglutinin group was significantly lower than that in the blank control group (p<0.01). This indicates that the fungus protein prepared in Example 1 has a significant effect on reducing the body weight of mice.

[0102] Table 6. Changes in blood glucose levels in mice at different drug administration times in each group.

[0103] ( ± SD, n=10, unit: mmol / L)

[0104] Group Blank control group Normal group Black fungus powder group Black fungus protein group Semaglutide group 0 weeks 13.8±0.8 8.6±0.9** 13.8±0.7 13.7±0.4 13.7±0.5 1 week 15.2±1.1 8.7±0.8** 13.4±0.6** 13.1±0.5** 13.3±0.4** 2 weeks 17.7±1.0 8.4±0.9** 12.3±0.6** 12.4±0.5** 12.0±0.5** 3 weeks 18.8±1.1 8.4±0.8** 12.0±0.6** 12.1±0.6** 11.6±0.4** 4 weeks 19.3±1.1 8.6±0.7** 11.7±0.6** 11.8±0.7** 11.4±0.4** 5 weeks 19.6±1.2 8.7±0.8** 11.5±0.6** 11.5±0.6** 11.1±0.4**

[0105] Note: Compared with the blank control group, * P < 0.05, ** P < 0.01

[0106] As can be seen from the results in Table 6, the blood glucose level of mice in the blank control group remained elevated. After administration of the test substance, the blood glucose levels of diabetic model mice in the black fungus powder group, black fungus protein group and smegglutinin group were significantly lower than those in the blank control group (p<0.01). It can be seen that the black fungus protein prepared in Example 1 has a significant effect on reducing blood glucose in mice, and the hypoglycemic effect of black fungus protein and black fungus powder is similar.

[0107] 2.5 Effects of purified black fungus protein on oral glucose tolerance test (OGTT) and insulin resistance test (ITT) in type II diabetic model mice

[0108] After the blood glucose effect experiment in the type II diabetes model mice in section 2.4, each group of mice was fasted for 12 hours and then administered 2 g / kg glucose by gavage. Blood glucose values ​​were measured at 0, 30, 60, 90 and 120 min to complete the oral glucose tolerance test (OGTT). The results of the OGTT are shown in Table 7.

[0109] Table 7 OGTT experiment in each group of mice

[0110] ( ± SD, n=10, unit: mmol / L)

[0111] Group Blank control group Normal group Black fungus powder group Black fungus protein group Semaglutide group 0 min 20.0±1.3 8.3±0.8** 11.3±0.6** 11.3±0.7** 10.8±0.4** 30 min 27.2±1.2 21.6±0.9** 22.1±1.1** 22.0±1.0** 21.0±0.6** 60 min 25.1±1.7 17.3±1.4** 19.4±1.3** 19.3±1.3** 19.3±1.2** 90 min 22.2±0.7 13.4±1.1** 17.7±1.1** 17.8±1.2** 17.4±0.8** 120 min 21.1±0.6 7.7±0.7** 11.2±0.5** 11.1±0.7** 10.6±0.4**

[0112] Note: Compared with the blank control group, * P < 0.05, ** P < 0.01

[0113] As shown in Table 7, compared with the normal group, the blood glucose level of the mice in the blank control group was significantly higher in the OGTT test (p<0.01). After administration, the glucose regulation ability of each test group was significantly improved compared with the blank control group (p<0.01), and the effect strength was: smegglutinin > black fungus protein ≈ black fungus powder.

[0114] Mice in each group who completed the oral glucose tolerance test (OGTT) were fasted for 4 hours and then injected intraperitoneally with 0.75 U / kg insulin. Blood glucose levels were measured at 0, 30, 60, 90 and 120 minutes to complete the insulin tolerance test (ITT). The results of the ITT are shown in Table 8.

[0115] Table 8 ITT test in mice of each group

[0116] ( ± SD, n=10, unit: mmol / L)

[0117] Group Blank control group Normal group Black fungus powder group Black fungus protein group Semaglutide group 0 min 23.2±0.8 7.9±0.4** 16.1±0.6** 15.9±0.6** 14.4±0.9** 30 min 17.1±1.1 6.4±0.4** 13.8±0.9** 13.5±0.8** 13.5±0.8** 60 min 21.5±1.0 5.9±0.3** 13.3±0.8** 12.9±0.7** 12.2±0.4** 90 min 22.9±0.7 6.2±0.3** 12.4±0.8** 12.1±0.6** 11.7±0.3** 120 min 23.4±1.1 7.4±0.4** 11.4±0.8** 11.2±0.9** 10.8±0.3**

[0118] Note: Compared with the blank control group, * P < 0.05, ** P < 0.01

[0119] As shown in Table 8, compared with the normal group, the blood glucose level in the blank control group was significantly higher (p<0.01). After administration, the insulin sensitivity of mice in each test group was significantly improved compared with the blank control group (p<0.01), with the effect strength being: smegglutinin > black fungus protein ≈ black fungus powder. Combining the results in Tables 5 and 6, it can be concluded that black fungus protein can significantly improve the glucose regulation ability and insulin sensitivity of diabetic mice, similar to the effect of black fungus itself, and possesses a good hypoglycemic effect.

[0120] Example 2

[0121] The blood sugar-lowering and antioxidant black fungus protein prepared in Example 1 was used to prepare functional products with blood sugar-lowering and antioxidant activities. These functional products include health supplements, pharmaceuticals, or functional oral products.

[0122] Example 3

[0123] The hypoglycemic and antioxidant black fungus protein prepared in Example 1 was used to prepare a hypoglycemic tablet product. The hypoglycemic tablet product is a black fungus protein compressed candy, each black fungus protein compressed candy weighing 0.8 g, containing 385 mg sorbitol, 200 mg resistant dextrin, 50 mg black fungus protein, 50 mg bitter melon peptide, 100 mg mulberry leaf powder and 15 mg magnesium stearate.

[0124] Example 4

[0125] The hypoglycemic and antioxidant black fungus protein prepared in Example 1 was used to prepare a hypoglycemic liquid product. The hypoglycemic liquid product is a black fungus protein anti-glycemic drink, each bottle containing 50 ml, comprising 37 g water, 6 g L-arabinose, 6 g fructooligosaccharides, 100 mg black fungus protein, 500 mg mulberry leaf powder, 200 mg green tea powder, 200 mg hawthorn powder, and 10 mg citric acid.

Claims

1. A black fungus protein with hypoglycemic and antioxidant properties, characterized in that: The amino acid sequence of the black fungus protein is shown in SEQ ID NO.

1.

2. The method for preparing a hypoglycemic and antioxidant black fungus protein as described in claim 1, characterized in that: Includes the following steps: S1. Pre-treatment of black fungus: Dry the black fungus, grind it into black fungus powder, sieve it, and store it for later use. S2. Extraction of black fungus protein: Add black fungus powder to NaOH solution, stir evenly, extract by ultrasonication in a 40℃ water bath, collect the supernatant by centrifugation, and obtain black fungus protein extract. S3. Purification of black fungus protein: The black fungus protein extract was filtered to remove impurities, subjected to ion exchange column chromatography, and each elution peak was collected separately. Each elution peak was freeze-dried, and the α-glucosidase inhibition rate and α-amylase inhibition rate were determined. The fraction with the highest α-glucosidase inhibition rate and α-amylase inhibition rate was selected to obtain the target purified fraction. S4. Secondary purification of black fungus protein: The target purified component prepared in S3 was dissolved in deionized water to prepare a target purified component solution, and purified by gel filtration chromatography. According to the elution curve results, different elution components were obtained and electrophoresed separately. The elution components with target protein bands were dialyzed and lyophilized to obtain purified black fungus protein.

3. The method for preparing a hypoglycemic and antioxidant black fungus protein according to claim 2, characterized in that: The black fungus in S1 is dried black fungus, which is placed in an electric heating drying oven and dried at a temperature of 55°C for 8 hours. The black fungus is then pulverized into black fungus powder using a functional pulverizer and sieved through a 100-mesh sieve.

4. The method for preparing a hypoglycemic and antioxidant black fungus protein according to claim 2, characterized in that: In step S2, 5g of black fungus powder was weighed and added to 0.007 M NaOH solution at a material-to-liquid ratio of 1:90 (m / V). The mixture was stirred until homogeneous. The mixture was then ultrasonically extracted at 100 W for 1.5 h and centrifuged at 8000 r / min for 20 min. The supernatant was collected.

5. The method for preparing a hypoglycemic and antioxidant black fungus protein according to claim 2, characterized in that: The ion exchange column chromatography in S3 is performed using a Q-Sepharose Fast Flow anion exchange column with a column volume of 80 ml, and filtration is carried out using filter paper.

6. The method for preparing a hypoglycemic and antioxidant black fungus protein according to claim 5, characterized in that: The chromatography in S3 includes the following steps: first, equilibrate the column with equilibration buffer; after equilibration, load the sample; then wash away unbound impurities on the column with equilibration buffer; perform linear gradient elution with 3-5 column bed volumes of eluent at 0-100% concentration; the equilibration buffer is 20 mM Na2HPO4-NaH2PO4 buffer with a pH of 5.8; the eluent is 1M NaCl + 20 mM Na2HPO4-NaH2PO4 buffer with a pH of 5.8; the equilibration and sample loading flow rates are 1.8 ml / min; the elution flow rate is 3.0 ml / min; the detection wavelength is 280 nm; and the chromatographic curve is recorded.

7. The method for preparing a hypoglycemic and antioxidant black fungus protein according to claim 2, characterized in that: The purification process in S4, which involves gel filtration chromatography, is replaced by purification using a Sephacryl S-100 gel chromatography column. The process includes the following steps: filtering and rinsing the Sephacryl S-100 gel medium to remove debris and impurities; packing the gel into a glass column, compacting it, eluting the column with 3-5 column volumes of deionized water, equilibrating the column with PBS buffer, loading the sample, eluting the column with PBS buffer, and recording the elution curve. The PBS buffer is a 50mM NaCl-20mM Na2HPO4-NaH2PO4 buffer with a pH of 6.

8. The elution flow rate is 0.6 ml / min, and the detection wavelength is 280 nm.

8. The method for preparing a hypoglycemic and antioxidant black fungus protein according to claim 2, characterized in that: In step S4, 200 mg of the target purified component prepared in step S3 is weighed out and dissolved in 2 ml of deionized water to prepare a 100 mg / ml target purified component solution.

9. The application of the hypoglycemic and antioxidant black fungus protein as described in claim 1 in the preparation of functional products with hypoglycemic and antioxidant activities.

10. The use of the hypoglycemic and antioxidant black fungus protein as described in claim 1 in the preparation of health products, drugs or functional oral products with hypoglycemic and antioxidant activities.

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