Pleurotus eryngii polypeptide as well as preparation method and application thereof

Abstract

The invention discloses a pleurotus eryngii polypeptide and a preparation method and application thereof, and the method comprises the following steps: step 1, adding pleurotus eryngii powder into deionized water to obtain a suspension A; adjusting the pH value of the suspension A to be alkaline, stirring for 2 hours at the temperature of 20-60 DEG C, centrifuging at the temperature of 4 DEG C, collecting supernate, washing precipitates for multiple times, and collecting the supernate; adjusting the pH value of the supernate to 4.3, centrifuging, collecting precipitate and washing to obtain a product B, adding deionized water into the product B to obtain a solution C, adjusting the pH value of the solution C to 7, dialyzing and freeze-drying to obtain pleurotus eryngii protein; step 2, dissolving the pleurotus eryngii protein in distilled water, and keeping the temperature under a water bath condition of 75 DEG C to obtain a pleurotus eryngii protein solution with the concentration of 21.45 mg / mL; alkaline protease is taken and added into the pleurotus eryngii protein solution, enzymolysis is conducted for 30-150 min, the water bath temperature is raised to 95 DEG C, enzymolysis inactivation, cooling and centrifugation are conducted, supernate is collected, freeze drying is conducted, and the pleurotus eryngii polypeptide is obtained and has good antioxidant activity and glycosyl resistance activity.

Description

Technical Field

[0001] The present invention relates to the extraction of polypeptides, in particular to a Pleurotus eryngii polypeptide and a preparation method and application thereof. Background Art

[0002] Many active substances in the human body exist in the form of peptides. Peptides are involved in the human body's hormones, nerves, cell growth and reproduction. Their importance lies in regulating the physiological functions of various systems and cells in the body, activating related enzyme systems in the body, promoting the permeability of intermediate metabolic membranes, or controlling DNA transcription or affecting specific protein synthesis, ultimately producing specific physiological effects. It can be seen that peptides play an extremely important role in the human body.

[0003] The human body produces a large number of free radicals during the metabolic process. Free radicals can seriously damage normal human cells through oxidation, and then induce human aging and disease. Polypeptides have extremely strong antioxidant properties and can effectively reduce the damage of free radicals to the human body. However, the content of natural antioxidant peptides in the human body is very small, far from enough to offset the damage of free radicals to human cells. Therefore, in order to delay human aging and reduce the probability of human disease, artificial extraction of antioxidant peptides has become a necessity.

[0004] In addition, with the rapid improvement of economic development level and changes in lifestyle, the high-sugar, high-fat diet structure and lack of exercise in the lifestyle have caused the incidence, mortality and medical costs of diabetes and its complications to show a rapid upward trend. Glycosylation inhibitors can significantly alleviate diabetes symptoms, delay the progression of diabetes and reduce the risk of diabetes complications. However, long-term use of such drugs is prone to cause a variety of toxic side effects. Therefore, how to extract natural advanced glycation end products (AGEs) inhibitors that are widely available, non-toxic and harmless, and easy to eat has attracted widespread attention.

[0005] As an edible fungus, Pleurotus eryngii is cultivated on a large scale in my country. The protein content in its dry product is as high as 21.44%, which is much higher than the protein content of dried products of shiitake mushrooms, white fungus and black fungus. It is an ideal raw material for artificial extraction of antioxidant peptides. In addition, the price of Pleurotus eryngii is relatively low. In particular, a large number of mushroom feet are produced during the production process, which are even cheaper and are usually treated as feed. Therefore, how to extract glycation end products (AGEs) inhibitors and antioxidant peptides from Pleurotus eryngii is of great practical significance. Summary of the invention

[0006] In view of the deficiencies in the prior art, the present invention provides a Pleurotus eryngii polypeptide and a preparation method and application thereof. The prepared Pleurotus eryngii polypeptide has good antioxidant activity and anti-glycosylation activity.

[0007] In order to achieve the above object, the present invention adopts the following technical solutions:

[0008] A method for preparing Pleurotus eryngii polypeptide comprises the following steps:

[0009] Step 1: Preparation of Pleurotus eryngii protein

[0010] Step 1.1, grind the dried Pleurotus eryngii into powder, add the Pleurotus eryngii powder into deionized water according to the ratio of 1g: (20-60mL) to obtain suspension A;

[0011] Step 1.2, first adjust the pH value of suspension A to 8-12 with alkali solution, then stir and extract at an extraction temperature of 20-60°C for 2h, then centrifuge at 4°C, collect the supernatant, wash the precipitate several times, and collect the supernatant in each centrifugal washing process, then mix all the collected supernatants for later use;

[0012] Step 1.3, adjusting the pH value of the supernatant collected in step 1.2 to an isoelectric point of 4.3 with acid solution, centrifuging, collecting and washing the precipitate to obtain product B;

[0013] Step 1.4, adding deionized water to product B to obtain solution C, adjusting the pH value of solution C to 7, dialyzing, and freeze-drying to obtain Pleurotus eryngii protein;

[0014] Step 2: Preparation of Pleurotus eryngii polypeptide

[0015] Step 2.1, dissolving Pleurotus eryngii protein in distilled water, and heating in a water bath at 75° C. to obtain a Pleurotus eryngii protein solution with a concentration of 21.45 mg / mL;

[0016] Step 2.2, under 75° C. water bath conditions, add alkaline protease to the Pleurotus eryngii protein solution according to 3% to 7% of the Pleurotus eryngii protein mass, and perform enzymolysis for 30 to 150 minutes. Then, the water bath temperature is increased to 95° C., enzymolysis is inactivated, and after cooling, centrifugation is performed, the supernatant is collected, and freeze-dried to obtain Pleurotus eryngii polypeptide.

[0017] Furthermore, the alkali solution in step 1.2 is a NaOH solution with a concentration of 2.0 mol / L.

[0018] Furthermore, the acid solution in step 1.3 is a HCl solution with a concentration of 2.0 mol / L.

[0019] Furthermore, the centrifugation in step 1.2 and step 1.3 is performed at a speed of 8000 r / min for 20 min.

[0020] Furthermore, the washing in step 1.2 and step 1.3 is performed by centrifugation washing with deionized water for 3 times.

[0021] Furthermore, the dialysis time of step 1.4 is 24 hours.

[0022] Furthermore, the centrifugation in step 2.2 is performed at a speed of 4000 r / min for 20 min.

[0023] A Pleurotus eryngii polypeptide.

[0024] Application of Pleurotus eryngii polypeptide as antioxidant peptide.

[0025] Application of Pleurotus eryngii polypeptide as AGEs inhibitor

[0026] Compared with the prior art, the present invention has the following technical effects:

[0027] The present invention first extracts Pleurotus eryngii protein from the inexpensive Pleurotus eryngii, and then extracts Pleurotus eryngii polypeptide from the Pleurotus eryngii protein. Not only is the process simple, but the prepared Pleurotus eryngii polypeptide has good antioxidant activity and can effectively reduce the oxidative damage of H2O2. At the same time, the extracted Pleurotus eryngii polypeptide has good anti-glycosylation activity and has a good inhibitory effect on fructosamine, dicarbonyl compounds and fluorescent AGEs, the products of the three stages in the non-enzymatic glycosylation process, and can effectively protect proteins from the attack of active carbonyls, thereby blocking the formation of AGEs. It has potential application prospects for the treatment of diabetes. It can be seen that the present invention provides theoretical guidance for the development of anti-aging and anti-diabetic dual-purpose medicine and food products. BRIEF DESCRIPTION OF THE DRAWINGS

[0028] Figure 1 : The protein extraction rate of Pleurotus eryngii of Examples 1 to 5 of the present invention;

[0029] Figure 2 : The protein extraction rate of Pleurotus eryngii of Examples 3, 6 to 9 of the present invention;

[0030] Figure 3 : The protein extraction rate of Pleurotus eryngii of Examples 8, 10 to 13 of the present invention;

[0031] Figure 4 : The degree of hydrolysis of the Pleurotus eryngii polypeptides of Examples 11 and 14 to 17 of the present invention;

[0032] Figure 5 : The degree of hydrolysis of the Pleurotus eryngii polypeptides of Examples 16 and 18 to 21 of the present invention;

[0033] Figure 6 : Statistical results of some peptide sequences in Pleurotus eryngii polypeptide prepared in Example 19 of the present invention;

[0034] Figure 7 : The effect of the Pleurotus eryngii polypeptide prepared in Example 19 of the present invention on the MDA content;

[0035] Figure 8 : The effect of the Pleurotus eryngii polypeptide prepared in Example 19 of the present invention on CAT enzyme activity;

[0036] Fig. 9 : The effect of the Pleurotus eryngii polypeptide prepared in Example 19 of the present invention on the intracellular TNF-α content;

[0037] Fig.10 , the effect of the Pleurotus eryngii polypeptide prepared in Example 19 of the present invention on the content of 1L-1β in cells;

[0038] Fig.11 , a bar graph showing the inhibition rate of fructosamine, the first-stage product of the non-enzymatic glycosylation process, by the Pleurotus eryngii polypeptide prepared in Example 19 of the present invention;

[0039] Fig.12 , a bar graph showing the inhibition rate of the Pleurotus eryngii polypeptide prepared in Example 19 of the present invention on dicarbonyl compounds, the second stage product in the non-enzymatic glycosylation process;

[0040] Fig.13 : A bar graph showing the inhibition rate of fluorescent AGEs, the third stage product of the non-enzymatic glycosylation process, by the Pleurotus eryngii polypeptide prepared in Example 19 of the present invention;

[0041] Fig.14 : Effect of the Pleurotus eryngii polypeptide prepared in Example 19 of the present invention on the carbonyl content. DETAILED DESCRIPTION

[0042] The specific contents of the present invention are further explained in detail below in conjunction with embodiments.

[0043] Example 1

[0044] Step 1: Preparation of Pleurotus eryngii protein

[0045] Step 1.1, grind the dried Pleurotus eryngii into powder, add the Pleurotus eryngii powder into deionized water at a ratio of 1 g: 20 mL, and obtain suspension A;

[0046] Step 1.2, first adjust the pH value of suspension A to 8 with a 2.0 mol / L NaOH solution, then stir and extract at an extraction temperature of 20°C for 2 h, then centrifuge at 8000 r / min at 4°C for 20 min, collect the supernatant, and at the same time, wash the precipitate with deionized water for 3 times, and collect the supernatant in each washing process, then mix all the collected supernatants for later use;

[0047] Step 1.3, adjusting the pH value of the supernatant collected in step 1.2 to an isoelectric point of 4.3 with a 2.0 mol / L HCl solution, centrifuging at a speed of 8000 r / min for 20 min, collecting the precipitate and washing it three times with deionized water to obtain product B;

[0048] Step 1.4, adding deionized water to product B to obtain solution C, adjusting the pH value of solution C to 7, dialyzing for 24 hours, and freeze-drying to obtain Pleurotus eryngii protein;

[0049] Step 2: Preparation of Pleurotus eryngii polypeptide

[0050] Step 2.1, dissolving Pleurotus eryngii protein in distilled water, and heating in a water bath at 75° C. to obtain a Pleurotus eryngii protein solution with a concentration of 21.45 mg / mL;

[0051] Step 2.2, under 75°C water bath conditions, add alkaline protease to the Pleurotus eryngii protein solution according to 3% of the Pleurotus eryngii protein mass, and enzymolyze for 30 minutes. Then, increase the water bath temperature to 95°C, inactivate the enzyme, cool, centrifuge at 4000r / min for 20 minutes, collect the supernatant, and freeze-dry to obtain Pleurotus eryngii polypeptide.

[0052] Example 2

[0053] The only difference from Example 1 is that: Step 1.1, grind the dried Pleurotus eryngii into powder, add the Pleurotus eryngii powder into deionized water at a ratio of 1 g: 30 mL, and obtain suspension A.

[0054] Example 3

[0055] The only difference from Example 1 is that in step 1.1, Pleurotus eryngii powder is added to deionized water in a ratio of 1 g:40 mL to obtain suspension A.

[0056] Example 4

[0057] The only difference from Example 1 is that in step 1.1, Pleurotus eryngii powder is added to deionized water in a ratio of 1 g:50 mL to obtain suspension A.

[0058] Example 5

[0059] The only difference from Example 1 is that in step 1.1, Pleurotus eryngii powder is added to deionized water at a ratio of 1 g: 60 mL to obtain suspension A.

[0060] Example 6

[0061] The only difference from Example 3 is that in step 1.2, the extraction is stirred at an extraction temperature of 30° C. for 2 h.

[0062] Example 7

[0063] The only difference from Example 3 is that in step 1.2, the extraction is stirred at an extraction temperature of 40° C. for 2 h.

[0064] Example 8

[0065] The only difference from Example 3 is that in step 1.2, the extraction is stirred at an extraction temperature of 50° C. for 2 h.

[0066] Example 9

[0067] The only difference from Example 3 is that in step 1.2, the extraction is stirred at an extraction temperature of 60° C. for 2 h.

[0068] Example 10

[0069] The only difference from Example 8 is that in step 1.2, the pH value of suspension A is adjusted to 9 using a 2.0 mol / L NaOH solution.

[0070] Embodiment 11

[0071] The only difference from Example 8 is that in step 1.2, the pH value of suspension A is adjusted to 10 using a 2.0 mol / L NaOH solution.

[0072] Example 12

[0073] The only difference from Example 8 is that in step 1.2, the pH value of suspension A is adjusted to 11 using a 2.0 mol / L NaOH solution.

[0074] Embodiment 13

[0075] The only difference from Example 8 is that in step 1.2, the pH value of suspension A is adjusted to 12 using a 2.0 mol / L NaOH solution.

[0076] Embodiment 14

[0077] The only difference from Example 11 is that in step 2.2, the enzymatic hydrolysis is carried out for 60 minutes.

[0078] Embodiment 15

[0079] The only difference from Example 11 is that in step 2.2, the enzymatic hydrolysis is carried out for 90 minutes.

[0080] Example 16

[0081] The only difference from Example 11 is that in step 2.2, the enzymatic hydrolysis is carried out for 120 min.

[0082] Embodiment 17

[0083] The only difference from Example 11 is that in step 2.2, the enzymatic hydrolysis is carried out for 180 min.

[0084] Embodiment 18

[0085] The only difference from Example 16 is that in step 2.2, alkaline protease is added to the Pleurotus eryngii protein solution according to 4% of the Pleurotus eryngii protein mass.

[0086] Embodiment 19

[0087] The only difference from Example 16 is that in step 2.2, alkaline protease is added to the Pleurotus eryngii protein solution according to 5% of the Pleurotus eryngii protein mass.

[0088] Embodiment 20

[0089] The only difference from Example 16 is that in step 2.2, alkaline protease is added to the Pleurotus eryngii protein solution according to 6% of the Pleurotus eryngii protein mass.

[0090] Embodiment 21

[0091] The only difference from Example 16 is that in step 2.2, alkaline protease is added to the Pleurotus eryngii protein solution according to 7% of the Pleurotus eryngii protein mass.

[0092] See also Figure 1 When the ratio of Pleurotus eryngii powder to deionized water is different, the yields of Pleurotus eryngii protein in Examples 1 to 5 are different, and when the ratio of Pleurotus eryngii powder to deionized water in Example 3 is 1g:50mL, the extraction rate of Pleurotus eryngii protein is the highest, indicating that 1g:50mL is the optimal material-liquid ratio.

[0093] See also Figure 2 At different extraction temperatures, the yields of Pleurotus eryngii protein in Example 3 and Examples 6 to 9 are different, and when the extraction temperature of Example 8 is 50°C, the extraction rate of Pleurotus eryngii protein is the highest, indicating that 50°C is the optimal extraction temperature.

[0094] See also Figure 3 When the pH value of the suspension A formed by mixing Pleurotus eryngii powder and deionized water is different, the yields of Pleurotus eryngii protein in Example 8 and Examples 10 to 13 are different, and when the pH value of Example 11 is 10, the extraction rate of Pleurotus eryngii protein is the highest, indicating that the optimal pH value is 10.

[0095] See also Figure 4Under different enzymatic hydrolysis times, the hydrolysis degrees of the Pleurotus eryngii polypeptides of Example 11 and Examples 14 to 17 are all different, and when the enzymatic hydrolysis time of Example 16 is 120 min, the hydrolysis degree of the Pleurotus eryngii polypeptide is the highest, indicating that the optimal enzymatic hydrolysis time is 120 min.

[0096] See also Figure 5 When the amount of alkaline protease added was different, the hydrolysis degree of the Pleurotus eryngii polypeptide of Example 16 and Examples 18 to 21 was different, and when the amount of alkaline protease added in Example 19 was 5% of the weight of the Pleurotus eryngii protein, the hydrolysis degree of the Pleurotus eryngii polypeptide was the highest.

[0097] In Example 19, the extraction rate of Pleurotus eryngii protein was (45±0.26)%, the molecular weight of Pleurotus eryngii protein was 68 kDa, and the extraction rate of Pleurotus eryngii polypeptide was (27.73±0.31)%.

[0098] The Pleurotus eryngii polypeptide prepared in Example 19 contains 1407 peptide sequences, among which the phenylalanine-aspartic acid-phenylalanine tripeptide (FNF) accounts for the highest proportion, reaching 2.47%. Figure 6 The partial peptide sequences of Pleurotus eryngii polypeptide extracted in Example 19 are shown.

[0099] The antioxidant activity of the Pleurotus eryngii polypeptide extracted in Example 1 was verified by in vitro oxidation indicators, including MDA content, CAT enzyme activity, intracellular TNF-α content, and intracellular 1L-1β content. The results are as follows: Figure 7 to Figure 10 As shown in the figure: light yellow represents the content of MDA, CAT, TNF-α or 1L-1β when H2O2 and Pleurotus eryngii polypeptide are not added; orange represents the content of MDA, CAT, TNF-α or 1L-1β when H2O2 is added but Pleurotus eryngii polypeptide is not added; three groups of blue represent the content of MDA, CAT, TNF-α or 1L-1β when Pleurotus eryngii polypeptide is added at different concentrations under the condition of H2O2; "***" and "###" both represent that the p value is less than 0.001; from Figure 7 to Figure 10 It can be seen that the Pleurotus eryngii polypeptide prepared in Example 1 can inhibit the oxidative stress induced by hydrogen peroxide and has antioxidant activity.

[0100] The BSA-Fru model was established to detect the inhibitory effect of the Pleurotus eryngii polypeptide extracted in Example 1 on anti-glycosylation products. The results are as follows: Figure 11 to Figure 13As shown, it can be seen that at different concentrations, the Pleurotus eryngii polypeptide extracted in Example 1 has a good inhibitory effect on the three-stage products of non-enzymatic glycosylation, fructosamine, dicarbonyl compounds and fluorescent AGEs, and as the concentration of Pleurotus eryngii polypeptide increases, the inhibitory effect on glycosamine, dicarbonyl compounds and fluorescent AGEs increases; at the same time, the effect of the Pleurotus eryngii polypeptide extracted in Example 1 on the protein functional group is also verified. The results are as follows Fig.14 As shown, it can be seen that after treatment with Pleurotus eryngii polypeptide, the carbonyl content was significantly reduced, and with the increase of Pleurotus eryngii polypeptide concentration, the carbonyl content became lower and lower. When the concentration of Pleurotus eryngii polypeptide reached 1000 μg / mL, the effect on the carbonyl content was close to the effect of positive drug aminoguanidine on the carbonyl content, which indicates that Pleurotus eryngii polypeptide can effectively protect proteins from the attack of active carbonyl groups, thereby blocking the formation of AGEs, and has potential application prospects for the treatment of diabetes.

Claims

1. A method for preparing Pleurotus eryngii polypeptide, characterized in that: The steps include: Step 1: Extraction of Pleurotus eryngii protein Step 1.1, grind the dried Pleurotus eryngii into powder, add the Pleurotus eryngii powder into deionized water according to the ratio of 1g: (20-60mL) to obtain suspension A; Step 1.2, first adjust the pH value of suspension A to 8-12 with alkali solution, then stir and extract at an extraction temperature of 20-60°C for 2h, then centrifuge at 4°C, collect the supernatant, wash the precipitate several times, and collect the supernatant in each centrifugal washing process, then mix all the collected supernatants for later use; Step 1.3, adjusting the pH value of the supernatant collected in step 1.2 to an isoelectric point of 4.3 with acid solution, centrifuging, collecting and washing the precipitate to obtain product B; Step 1.4, adding deionized water to product B to obtain solution C, adjusting the pH value of solution C to 7, dialyzing, and freeze-drying to obtain Pleurotus eryngii protein; Step 2: Extraction of Pleurotus eryngii peptides Step 2.1, dissolving Pleurotus eryngii protein in distilled water, and heating in a water bath at 75° C. to obtain a Pleurotus eryngii protein solution with a concentration of 21.45 mg / mL; Step 2.2, under 75° C. water bath conditions, add alkaline protease to the Pleurotus eryngii protein solution according to 3% to 7% of the Pleurotus eryngii protein mass, and perform enzymolysis for 30 to 150 minutes. Then, the water bath temperature is increased to 95° C., enzymolysis is inactivated, and after cooling, centrifugation is performed, the supernatant is collected, and freeze-dried to obtain Pleurotus eryngii polypeptide.

2. The method for preparing Pleurotus eryngii polypeptide according to claim 1, characterized in that: The alkali solution in step 1.2 is a NaOH solution with a concentration of 2.0 mol / L.

3. The method for preparing Pleurotus eryngii polypeptide according to claim 1, characterized in that: The acid solution in step 1.3 is a HCl solution with a concentration of 2.0 mol / L.

4. The method for preparing Pleurotus eryngii polypeptide according to claim 1, characterized in that: The centrifugation in step 1.2 and step 1.3 is performed at a speed of 8000 r / min for 20 min.

5. The method for preparing Pleurotus eryngii polypeptide according to claim 1, characterized in that: The washing in step 1.2 and step 1.3 is performed by centrifugation washing with deionized water three times.

6. The method for preparing Pleurotus eryngii polypeptide according to claim 1, characterized in that: The dialysis time in step 1.4 is 24 hours.

7. The method for preparing Pleurotus eryngii polypeptide according to claim 1, characterized in that: The centrifugation in step 2.2 is performed at a speed of 4000 r / min for 20 min.

8. A Pleurotus eryngii polypeptide prepared by the method according to any one of claims 1 to 7.

9. A use of the Pleurotus eryngii polypeptide as claimed in claim 8 as an antioxidant peptide.

10. Use of the Pleurotus eryngii polypeptide as claimed in claim 8 as an AGEs inhibitor.

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