A method for preparing protein freeze-dried powder, polypeptide freeze-dried powder and amino acid freeze-dried powder from Dictyophora rubrozonata

Abstract

This invention provides a method for preparing freeze-dried protein powder, freeze-dried peptide powder, and freeze-dried amino acid powder from *Dictyophora indicum*, relating to the field of *Dictyophora indicum* extraction technology. Using *Dictyophora indicum* as raw material, this invention employs a combination of organic solvent soaking, alkaline precipitation, and enzymatic hydrolysis, followed by centrifugation and freeze-drying processes, to obtain freeze-dried protein powder, freeze-dried peptide powder, and freeze-dried amino acid powder from *Dictyophora indicum*. The extraction method of this invention is simple and significantly improves extraction efficiency.

Description

Technical Field

[0001] This invention relates to the field of protein extraction technology, and in particular to a method for preparing freeze-dried protein powder, freeze-dried polypeptide powder and freeze-dried amino acid powder from *Dictyophora indica*. Background Technology

[0002] Bamboo fungus (Dictyophora indusiata (Vent. ex Pers) Fisch.) is a fungus belonging to the genus Dictyophora in the family Phallaceae. Also known as bamboo shoots or bamboo ginseng, it is a cryptogamic fungus that parasitizes the roots of dead bamboo. There are four common and edible species of bamboo fungus: long-skirted bamboo fungus, short-skirted bamboo fungus, spiny-topped bamboo fungus, and red-topped bamboo fungus. Dried bamboo fungus contains up to 37.94% crude protein, 35.41% total sugar, 4.74% crude fiber, 4.98% crude fat, and 10.56% ash. This demonstrates that bamboo fungus contains abundant protein and amino acids, surpassing those found in vegetables and fruits. Furthermore, most of the amino acids are present in the form of fungal protein, making them less prone to loss. Therefore, bamboo fungus can serve as a good source of protein.

[0003] However, due to the cellular characteristics of bamboo fungus, its protein extraction rate is relatively low, and consuming small amounts of bamboo fungus cannot effectively achieve the goal of ingesting high-quality protein. Therefore, how to efficiently extract bamboo fungus protein and improve its utilization rate by the human body is one of the technical problems that needs to be solved. Summary of the Invention

[0004] To address the aforementioned issues, this invention utilizes *Dictyophora indicum* as raw material, combining methods such as organic solvent soaking, alkaline precipitation, and enzymatic hydrolysis. Through processes including centrifugation and freeze-drying, it achieves efficient extraction of proteins, polysaccharides, and amino acids from *Dictyophora indicum*.

[0005] The method for preparing protein freeze-dried powder, polypeptide freeze-dried powder and amino acid freeze-dried powder from *Dictyophora indica* according to the present invention includes the following steps:

[0006] (1) Wash the fresh red-topped bamboo fungus, drain the water and make it into a paste;

[0007] (2) The homogenate and the degreasing agent are mixed at a mass ratio of 20:1 to degrease the homogenate;

[0008] (3) Adjust the pH of the degreased homogenate to 9-11;

[0009] (4) The homogenate after step (3) is subjected to ultrasonic treatment, and the supernatant is taken after centrifugation;

[0010] (5) Adjust the pH of the supernatant to 2.0-5.5, allow it to stand for acid precipitation, and collect the precipitated solid after centrifugation;

[0011] (6) The precipitated solid described in step (5) is subjected to vacuum freeze-drying to obtain freeze-dried protein powder of red-topped bamboo fungus;

[0012] (7) The precipitated solid described in step (5) is mixed with distilled water at a ratio of 1g:50mL. After being dissolved by ultrasonication, alkaline protease is added and the mixture is heated in a water bath at 55℃ for 3 hours for enzymatic hydrolysis. After heating, the enzyme is inactivated and then concentrated. The concentrated solution is freeze-dried to obtain red-topped bamboo fungus stalk polypeptide.

[0013] (8) The precipitated solid described in step (5) is mixed with 6 mol / L HCl solution at a ratio of 1 g / mL, hydrolyzed at 110℃ for 22 h, filtered, concentrated, and freeze-dried to obtain red-topped bamboo fungus amino acid freeze-dried powder.

[0014] The HCl solution contained 0.1% (w / v) phenol.

[0015] Furthermore, in step (2), the degreasing agent is petroleum ether, and the stirring and mixing time is 10-30 hours.

[0016] Further, step (3) involves adjusting the pH value of the degreased homogenate using sodium hydroxide solution.

[0017] Furthermore, in step (4), the ultrasonic power is 330-550W, the ultrasonic temperature is 45℃, and the ultrasonic time is 10-30min.

[0018] Furthermore, in step (4), the centrifugation speed is 5000 r / min and the centrifugation time is 20 min.

[0019] Further, step (5) involves adjusting the pH of the supernatant using hydrochloric acid, wherein the concentration of the hydrochloric acid is 1 mol / L.

[0020] Furthermore, in step (5), the centrifugation speed is 5000 r / min and the centrifugation time is 25 min.

[0021] Furthermore, the enzyme inactivation temperature in step (7) is 95°C and the enzyme inactivation time is 10 min.

[0022] Furthermore, the alkaline protease described in step (7) is added with an enzyme activity of 3000 U.

[0023] Compared with the prior art, the beneficial technical effects of the present invention are as follows:

[0024] The extraction method of red-topped bamboo fungus protein of the present invention is quick and easy to operate, with a short experimental cycle, and the obtained red-topped bamboo fungus protein, polypeptides and amino acids have good biological activity and human utilization rate. Attached Figure Description

[0025] The present invention will be further described below with reference to the accompanying drawings.

[0026] Figure 1 To investigate the effect of different extraction conditions on the protein extraction rate of *Dictyophora indica*. Detailed Implementation

[0027] The technical solution provided by the present invention will be further described below with reference to the embodiments.

[0028] Example 1

[0029] A single-factor controlled experiment was conducted using defatted red-topped bamboo fungus homogenate as raw material to test various conditions.

[0030] The preparation process of the defatted red-topped bamboo fungus homogenate is as follows: wash the fresh red-topped bamboo fungus volva, drain the water, and put it into a blender to make a homogenate; use petroleum ether as a defatting agent and perform defatting treatment for 24 hours at a material-to-liquid ratio of 1:20 (g / mL) to obtain the defatted red-topped bamboo fungus homogenate.

[0031] (1) Optimize the feed-liquid ratio:

[0032] Add a 30% sodium hydroxide solution to the defatted red-topped bamboo fungus homogenate, and add a pH adjuster (1 mol / L HCl and / or 1 mol / L NaOH) to adjust the pH to 4. After ultrasonic extraction at 500W and 45℃ for 30 min, centrifuge at 5000 r / min for 20 min, take the supernatant, adjust the pH to 2 with 1 mol / L HCl solution for acid precipitation, centrifuge at 5000 r / min for 25 min after acid precipitation, and freeze-dry the precipitate under vacuum to obtain the red-topped bamboo fungus volva protein.

[0033] Depend on Figure 1 As shown in D, with the increase of the solid-liquid ratio, the solvent is continuously increased, and the solute is fully dispersed, greatly increasing the contact area between the protein in *Dictyophora indica* volva and the solvent, thus promoting protein dissolution. The protein extraction rate reaches its maximum (2.86%) at a ratio of 1:20. Further increasing the solvent dosage leads to a gradual plateau in the extraction rate. Furthermore, considering that excessive solvent dosage would increase the economic cost of post-processing, a solid-liquid ratio of 1:20 (g / mL) is more suitable.

[0034] (2) pH optimization:

[0035] Add a 30% sodium hydroxide solution to the defatted red-topped bamboo fungus homogenate at a material-to-liquid ratio of 1:20. Adjust the pH value with a pH adjuster. After ultrasonic extraction at 500W and 45℃ for 30 min, centrifuge at 5000r / min for 20 min. Take the supernatant and adjust the pH value to 2 with 1mol / L HCl solution for acid precipitation. After acid precipitation, centrifuge at 5000r / min for 25 min. After vacuum freeze-drying, obtain the red-topped bamboo fungus volva protein.

[0036] Depend on Figure 1 As shown in A, the protein yield increases with increasing solution pH. However, the closer the pH is to the isoelectric point, the lower the protein solubility, ultimately leading to a decrease in protein yield. At pH values ​​above the isoelectric point, a larger net negative charge is generated. Due to the repulsion of like charges, the protein becomes more dispersed, thus increasing the protein yield.

[0037] (3) Optimize ultrasonic power:

[0038] Add a 30% sodium hydroxide solution to the defatted red-topped bamboo fungus homogenate at a material-to-liquid ratio of 1:20. Adjust the pH to 4 with a pH adjuster. After ultrasonic extraction at 45°C for 30 min under specific power, centrifuge at 5000 r / min for 20 min. Take the supernatant and adjust the pH to 2 with 1 mol / L HCl solution for acid precipitation. After acid precipitation, centrifuge at 5000 r / min for 25 min. After vacuum freeze-drying, obtain the red-topped bamboo fungus volva protein.

[0039] Depend on Figure 1 As shown in C, the protein yield initially increases and then decreases with increasing ultrasonic power. The cavitation and mechanical effects of ultrasound disrupt the quaternary structure of proteins, causing the release of subunits from small protein molecules. Some polar amino acid residues migrate to the outside of the protein molecule, enhancing the interaction between the protein and water molecules and increasing solubility. The protein yield reaches its maximum value of 3.02% in the reaction system when the ultrasonic power exceeds 495W.

[0040] (4) Optimize extraction time:

[0041] Add a 30% sodium hydroxide solution to the defatted red-topped bamboo fungus homogenate at a material-to-liquid ratio of 1:20. Adjust the pH to 4 with a pH adjuster. After ultrasonic extraction at 500W and 45℃ for a specific time, centrifuge at 5000r / min for 20min. Take the supernatant and adjust the pH to 2 with 1mol / L HCl solution for acid precipitation. After acid precipitation, centrifuge at 5000r / min for 25min. The precipitate is then freeze-dried under vacuum to obtain the red-topped bamboo fungus volva protein.

[0042] Depend on Figure 1As shown in Figure B, the protein yield initially increases and then decreases with increasing extraction time. The protein extraction rate from *Dictyophora indica* 'Red-topped' continuously increases with prolonged extraction time. At 25 minutes, the extraction rate reaches its maximum of 2.8%, and further extension of the extraction time leads to a decrease in the extraction rate. This may be due to the interaction of precipitated proteins during prolonged extraction, resulting in flocculation and precipitation. Therefore, an extraction time of 25 minutes is considered optimal.

[0043] The results showed that under the conditions of extraction pH=11, ultrasonic power of 550W, material-liquid ratio of 1:20 (g / mL) and extraction time of 25 min, a high extraction rate of red-topped bamboo fungus protein can be obtained without consuming too much time, thus reducing material costs.

[0044] Example 2

[0045] A method for extracting protein from *Dictyophora indica*, comprising the following steps:

[0046] (1) Wash the fresh red-topped bamboo fungus, drain the water, and put it into a blender to make a homogenous paste.

[0047] (2) Use petroleum ether as a degreasing agent at a ratio of 1:20 (g / mL) and degrease for 24 hours;

[0048] (3) Add a 30% sodium hydroxide solution at a material-to-liquid ratio of 1:5, and adjust the pH to 11 with 1 mol / L HCl and NaOH solutions;

[0049] (4) After ultrasonic heating and extraction at 45°C for 25 min under ultrasonic 550W conditions, centrifuge at 5000r / min for 20 min and take the supernatant.

[0050] (5) Adjust the pH to 2.5 with 1 mol / L HCl solution for acid precipitation, centrifuge at 5000 r / min for 25 min, and collect the precipitate;

[0051] (6) Vacuum freeze drying was performed to obtain freeze-dried protein powder of *Dictyophora indica*.

[0052] The anticancer activity of the red-topped bamboo fungus protein obtained in Example 2 was tested:

[0053] The detection method is as follows: A549, HELA, K562, MDA-231, and PC3 cancer cells were selected as screening samples for the anticancer activity of *Dictyophora indica* protein. The protein concentrations were set at 30 μg / mL, 60 μg / mL, 90 μg / mL, 120 μg / mL, and 150 μg / mL. The detection results are as follows:

[0054] Concentration (μg / ml) A549 HELA K562 MDA-231 PC3 30 26.79％ 20.81％ 19.84％ 13.21％ 13.77％ 60 23.46％ 30.20％ 22.61％ 20.48％ 20.13％ 90 21.64％ 30.88％ 23.27％ 22.82％ 19.35％ 120 23.99％ 36.62％ 31.59％ 24.81％ 14.00％ 150 38.59％ 40.47％ 38.85％ 27.79％ 30.55％

[0055] The results showed that the red-topped bamboo fungus protein prepared in this invention had significant inhibitory activity against A549, HELA, K562, MDA-231, and PC3 cancer cells.

[0056] Example 3

[0057] A method for extracting polypeptides from *Dictyophora indicum*, comprising the following steps:

[0058] (1) Wash the fresh red-topped bamboo fungus, drain the water, and put it into a blender to make a homogenous paste.

[0059] (2) Use petroleum ether as a degreasing agent with an addition ratio of 1:20 (g / mL) and degrease for 24 hours;

[0060] (3) Add a 30% sodium hydroxide solution at a material-to-liquid ratio of 1:5, and adjust the pH to 11 with 1 mol / L HCl and NaOH solutions;

[0061] (4) After ultrasonic heating and extraction at 45°C for 25 min under ultrasonic 550W conditions, centrifuge at 5000r / min for 20 min and take the supernatant.

[0062] (5) Adjust the pH to 2.5 with 1 mol / L HCl solution for acid precipitation, centrifuge at 5000 r / min for 25 min, and collect the precipitate;

[0063] (6) Take the precipitate from step (5) and add it to distilled water at a ratio of 1:50. Sonicate at 550W for 10 minutes to dissolve it. Add alkaline protease with an enzyme activity of 3000U and heat in a water bath at 55℃ for 3 hours. After heating, remove the sample and inactivate the enzyme in a water bath at 95℃ for 10 minutes. Then transfer the solution to a flask and concentrate it using a rotary evaporator. Take the concentrated sample and freeze-dry it under vacuum to obtain the red-topped bamboo fungus stigma polypeptide.

[0064] The anticancer activity of the red-topped bamboo fungus polypeptide prepared in Example 2 was tested:

[0065] The detection method is as follows: A549, HELA, K562, MDA-231, and PC3 cancer cells were selected as screening samples for the anticancer activity of *Dictyophora indica* protein. Concentrations of 30 μg / mL, 60 μg / mL, 90 μg / mL, 120 μg / mL, and 150 μg / mL were set. The detection results are as follows:

[0066]

[0067]

[0068] The results showed that the red-topped bamboo fungus protein prepared in this invention had significant inhibitory activity against A549, HELA, K562, MDA-231, and PC3 cancer cells.

[0069] Example 3

[0070] A method for extracting amino acids from *Dictyophora indica*, comprising the following steps:

[0071] (1) Wash the fresh red-topped bamboo fungus, drain the water, and put it into a blender to make a homogenous paste.

[0072] (2) Use petroleum ether as a degreasing agent with an addition ratio of 1:20 (g / mL) and degrease for 24 hours;

[0073] (3) Add a 30% sodium hydroxide solution at a material-to-liquid ratio of 1:5, and adjust the pH to 11 with 1 mol / L HCl and NaOH solutions;

[0074] (4) After ultrasonic heating and extraction at 45°C for 25 min under ultrasonic 550W conditions, centrifuge at 5000r / min for 20 min and take the supernatant.

[0075] (5) Adjust the pH to 2.5 with 1 mol / L HCl solution for acid precipitation, centrifuge at 5000 r / min for 25 min, and collect the precipitate;

[0076] (6) The precipitate from step (5) was hydrolyzed in 6 mol / L HCl (containing 0.1% (w / v) phenol) at 110°C for 22 h. The hydrolysis product was filtered with qualitative filter paper, concentrated, and freeze-dried to obtain amino acids.

[0077] 10 mg of the lyophilized amino acid powder was accurately dissolved in 1 mL of distilled water. The sample solution was filtered through a 0.45 μm filter membrane, and the amino acid composition was determined using an amino acid analyzer (S-433D). The results are as follows:

[0078]

[0079] This document uses specific examples to illustrate the principles and implementation methods of the present invention. The descriptions of the above embodiments are only for the purpose of helping to understand the method and core ideas of the present invention. Furthermore, those skilled in the art will recognize that, based on the ideas of the present invention, there will be changes in the specific implementation methods and application scope. Therefore, the content of this specification should not be construed as a limitation of the present invention.

Claims

1. The application of a *Dictyophora indica* protein or polypeptide in inhibiting A549, HELA, K562, MDA-231, and PC3 cancer cells, characterized in that... The method for preparing the protein or polypeptide from *Dictyophora indica* includes the following steps: (1) Wash the fresh red-topped bamboo fungus, drain the water and make it into a paste; (2) The homogenate and the degreasing agent are mixed at a mass ratio of 20:1 to degrease the homogenate; (3) Adjust the pH of the degreased homogenate to 11; (4) The homogenate after step (3) is ultrasonicated at 550 W for 25 min, and the supernatant is taken after centrifugation. (5) Adjust the pH of the supernatant to 2.0~5.5, let it stand for acid precipitation, and collect the precipitated solid after centrifugation; (6) The precipitated solid described in step (5) is subjected to vacuum freeze-drying to obtain freeze-dried protein powder of red-topped bamboo fungus; (7) The precipitated solid described in step (5) is mixed with distilled water at a ratio of 1g:50mL. After being dissolved by ultrasonication, alkaline protease is added and the mixture is heated in a water bath at 55℃ for 3 hours for enzymatic hydrolysis. After heating, the enzyme is inactivated and then concentrated. The concentrated solution is freeze-dried to obtain red-topped bamboo fungus stalk polypeptide. (8) The precipitated solid described in step (5) is mixed with 6 mol / L HCl solution at a ratio of 1 g / mL, hydrolyzed at 110℃ for 22 h, filtered, concentrated, and freeze-dried to obtain red-topped bamboo fungus amino acid freeze-dried powder. The HCl solution contained 0.1% (w / v) phenol.

2. The method for preparing protein freeze-dried powder, polypeptide freeze-dried powder, and amino acid freeze-dried powder from *Dictyophora indicum* according to claim 1, characterized in that, In step (2), the degreasing agent is petroleum ether, and the stirring time is 10-30 h.

3. The application according to claim 1, characterized in that, Step (3) involves adjusting the pH value of the degreased homogenate using sodium hydroxide solution.

4. The application according to claim 1, characterized in that, In step (4), the centrifugation speed is 5000 r / min and the centrifugation time is 20 min.

5. The application according to claim 1, characterized in that, Step (5) involves adjusting the pH of the supernatant using hydrochloric acid, wherein the concentration of the hydrochloric acid is 1 mol / L.

6. The application according to claim 1, characterized in that, The centrifugation speed in step (5) is 5000 r / min and the centrifugation time is 25 min.

7. The application characteristic according to claim 1, The enzyme inactivation temperature in step (7) is 95℃ and the enzyme inactivation time is 10 min.

8. The application according to claim 1, characterized in that, The alkaline protease in step (7) is added at an enzyme activity of 3000 U.

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