Cordyceps militaris mycelium material as well as preparation method and application thereof

Abstract

The invention provides a cordyceps militaris mycelium and a preparation method and application thereof, and relates to the technical field of biological materials. According to the invention, chitinase is added into the cordyceps militaris mycelium material to carry out enzymolysis on chitin to obtain chitooligosaccharide and N-acetylglucosamine, so that beta-glucan in a mycelium framework is dissolved out, and the grafting efficiency of the cordyceps militaris mycelium material is improved; furthermore, the antibacterial effect of the antibacterial material is improved by carrying out grafting reaction on the cordyceps militaris mycelium material and an exogenous monomer antibacterial peptide which has antibacterial performance and contains carboxyl groups.

Description

Technical Field

[0001] The invention relates to the technical field of biomaterials, and in particular to a Cordyceps militaris mycelium material and a preparation method and application thereof. Background Art

[0002] In recent years, mycelium materials have attracted widespread attention in the field of scientific research. The environmental protection, sustainability and diversity of mycelium materials have made them have broad application prospects in various fields such as medicine, packaging, and construction. Fungal mycelium is a natural, renewable and valuable cell structure. Its cell wall is mainly composed of glucan, protein, and chitin. Among them, chitin as the structural basis skeleton accounts for most of the total content of mycelium. β-glucan, mannan and some glycoproteins as the matrix of mycelium cell wall are like fillers on the skeleton, combined with chitin to strengthen the strength of the cell wall.

[0003] The grafting reaction of mycelium materials refers to the process of connecting monomers with specific functions to the surface or inside of mycelium materials through chemical reactions, thereby giving mycelium materials new properties and functions. At present, the preparation of antibacterial mycelium materials is mostly done by adding antibacterial agents from the outside through grafting reactions to improve the antibacterial properties of mycelium materials. However, this method has technical problems such as insufficient grafting dosage, low content of antibacterial agents added, inability to effectively inhibit or kill pathogens, and the antibacterial properties of the materials do not achieve the expected effect. Summary of the invention The present invention provides a Cordyceps militaris mycelium material and a preparation method and application thereof, which is obtained by adding chitinase to the Cordyceps militaris mycelium material for enzymatic hydrolysis. The Cordyceps militaris mycelium material is subjected to chitin enzymatic hydrolysis to hydrolyze chitosan oligosaccharides and N-acetylglucosamine, so that β-glucan in the mycelium framework is dissolved, thereby increasing the grafting efficiency of the Cordyceps militaris mycelium material, and further by grafting the Cordyceps militaris mycelium material with an exogenous monomer containing a carboxyl group having antibacterial properties to obtain an antibacterial mycelium material with improved antibacterial effect.

[0004] In one aspect, the present invention provides a Cordyceps militaris mycelium material and a preparation method thereof, comprising the following steps: (1) inoculating a Cordyceps militaris spore suspension into a potato glucose water culture medium containing yeast extract and glycerol, filtering and washing to obtain bacterial balls, placing the filtered bacterial balls into a culture dish, and drying to obtain a Cordyceps militaris mycelium material precursor; (2) Adding a chitinase solution to the Cordyceps militaris mycelium material precursor obtained in step (1) for enzymatic hydrolysis to obtain a Cordyceps militaris mycelium material.

[0005] Furthermore, the culture temperature of the Cordyceps militaris spore suspension in the above step (1) is 24°C-30°C, the pH is 5-8, and the culture time is 60-80 hours; and the drying time is 4-6 hours.

[0006] Furthermore, in the potato glucose water culture medium in step (1), the amount of yeast extract added is 2% to 8%, and the amount of glycerol added is 4% to 8%.

[0007] Furthermore, the preparation method of the potato glucose water culture medium described in the above step (1) is to weigh corn flour: glucose: water in a ratio of 2:1:10, stir evenly and put into a sterilizer at 115-120°C for about 25-30 minutes.

[0008] Furthermore, the concentration of the chitinase solution in the above step (2) is 0.4-0.6 g / mL, and the added amount is 1 mL per 1 g of Cordyceps militaris mycelium material.

[0009] Furthermore, the enzymatic hydrolysis time in the above step (2) is 0.5-1 h, and the temperature is 26-30°C.

[0010] Furthermore, the Cordyceps militaris mycelium material in the above step (2) is further subjected to a grafting reaction with a monomer having an antibacterial function.

[0011] On the other hand, the present invention provides a Cordyceps militaris mycelium material prepared by the above method.

[0012] In another aspect, the present invention provides a use of the Cordyceps militaris mycelium material prepared by the above method in preparing an antibacterial mycelium material.

[0013] Furthermore, the above application is to graft the Cordyceps militaris mycelium material prepared by the above method with an exogenous monomer containing a carboxyl group having antibacterial properties to obtain an antibacterial mycelium material.

[0014] Furthermore, the above-mentioned monomers containing carboxyl groups having antibacterial properties include antimicrobial peptides, carboxypenicillins, and β-lactam antibiotics except monocyclic β-lactams.

[0015] Compared with the prior art, the technical solution of the present invention has the following advantages: (1) The present invention uses chitinase enzymatic hydrolysis to dissolve the chitin in the mycelium framework, thereby dissolving the β-glucan in the content, increasing the β-glucan content on the mycelium surface by about 12%. By exposing the effective groups, the grafting efficiency between the mycelium material and the exogenous monomer with antibacterial properties is increased, thereby exerting the antibacterial properties of the mycelium material to a greater extent. Figure 7As shown, the antibacterial performance of the antibacterial mycelium material of the present invention prepared by chitinase hydrolysis is improved by 420% compared with the antibacterial effect of the antibacterial mycelium material prepared without chitinase hydrolysis.

[0016] (2) In the present invention, during the cultivation process of Cordyceps militaris, by adding 2%-8% yeast extract and 4%-8% glycerol to the culture medium, the content of β-glucan, an effective ingredient in the Cordyceps militaris pellets, can be increased by 4.1%. BRIEF DESCRIPTION OF THE DRAWINGS

[0017] Figure 1 Schematic diagram of the mycelial cell wall components; Figure 2 The fluorescence intensity data of the hyphae cell wall before and after being enzymatically hydrolyzed by chitinase; Figure 3 The fluorescence intensity data under different culture temperatures (°C) are shown; Figure 4 It is the fluorescence intensity data graph under different culture pH conditions; Figure 5 The fluorescence intensity data graph under different glycerol addition amounts (%); Figure 6 The fluorescence intensity data graph is shown under different yeast extract addition amounts (%); Figure 7 This is a comparison chart of the antibacterial effect of grafted antimicrobial peptide materials on plate culture; Figure 8 Schematic diagram of the number of single colonies of grafted antimicrobial peptide materials in plate culture. DETAILED DESCRIPTION

[0018] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings required for use in the embodiments or the description of the prior art will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying creative labor.

[0019] In one embodiment of the present invention, a method for preparing a Cordyceps militaris mycelium material is provided, comprising the following steps: (1) inoculating a Cordyceps militaris spore suspension into a potato glucose water culture medium containing yeast extract and glycerol for cultivation, filtering and washing to obtain bacterial balls, placing the filtered bacterial balls in a culture dish and drying them to obtain a Cordyceps militaris mycelium material precursor; (2) Take the Cordyceps militaris mycelium material obtained in step (1), add chitinase solution to perform enzymolysis, and obtain the Cordyceps militaris mycelium material.

[0020] In step (1), the Cordyceps militaris spore suspension is prepared by the following method: The frozen and preserved Cordyceps militaris strain is activated and cultured on a potato solid culture medium to obtain a first-generation Cordyceps militaris strain, which is then subcultured to obtain a second-generation Cordyceps militaris strain, and then washed and filtered to obtain a Cordyceps militaris spore suspension.

[0021] Applicable culture conditions are: culture temperature of 26-30°C, for example, 26-27°C, 27-28°C, 28-29°C, 29-30°C; culture time of 6-8 days.

[0022] The culture temperature of the Cordyceps militaris spore suspension in step (1) is 24-30°C, for example, 24-26°C, 26-28°C, or 28-30°C.

[0023] The culture pH of the Cordyceps militaris spore suspension in step (1) is 5-8, for example, 5-6, 6-7, or 7-8.

[0024] The culturing time of the Cordyceps militaris spore suspension in step (1) is 60-80 h, for example, 60-65 h, 65-70 h, 70-75 h, or 75-80 h.

[0025] In step (1) of the potato glucose water culture medium, the amount of yeast extract added is 2-8%, for example, 2-4%, 4-6%, or 6-8%; the amount of glycerol added is 4-8%, for example, 4-6%, or 6-8%.

[0026] The preparation method of the potato glucose water culture medium in step (1) is to weigh corn flour: glucose: water in a ratio of 2:1:10, stir evenly and put into a sterilizer at 115-120°C for 25-30 minutes.

[0027] In step (1), the bacterial pellets are placed in the culture dish and dried for 4-6 hours, for example, 4-5 hours or 5-6 hours.

[0028] The concentration of the chitinase solution in step (2) is 0.4-0.6 g / mL, and the added amount is 1 mL per 1 g of Cordyceps militaris mycelium material.

[0029] In step (2), the chitinase solution is added for enzymolysis for 0.5 to 1 h at a temperature of 26 to 30°C.

[0030] The Cordyceps militaris mycelium material obtained in step (2) can be further subjected to a grafting reaction with a monomer having an antibacterial function.

[0031] An embodiment of the present invention provides a Cordyceps militaris mycelium material, which is obtained by adding chitinase to the Cordyceps militaris mycelium material for enzymolysis.

[0032] The cell wall of fungal hyphae is mainly composed of glucan, protein and chitin, among which chitin as the basic structural skeleton accounts for most of the total content of mycelium. β-glucan, mannan and some glycoproteins as the matrix of mycelium cell wall are like fillers on the skeleton, combined with chitin to strengthen the strength of the cell wall. Chitin as the basic structural skeleton is usually highly crystalline and has poor solubility, which limits the grafting reaction between mycelium material and grafting reagent.

[0033] The grafting reaction is a chemical reaction that introduces branches into a polymer chain, specifically, a segment of a monomer or oligomer is chemically bonded to the main chain of another polymer chain to form a graft copolymer with a branched structure.

[0034] The chitin of the Cordyceps militaris mycelium material that has been enzymatically hydrolyzed by chitin is enzymatically hydrolyzed into chitooligosaccharides and N-acetylglucosamine, which dissolves the β-glucan in the mycelium framework, thereby increasing the grafting efficiency of the Cordyceps militaris mycelium material. The antibacterial mycelium material with improved antibacterial properties can be further obtained by grafting the Cordyceps militaris mycelium material with exogenous carboxyl group-containing monomers having antibacterial properties.

[0035] Another embodiment of the present invention provides an application of a Cordyceps militaris mycelium material in the preparation of an antibacterial mycelium material, wherein the antibacterial mycelium material is obtained by grafting the Cordyceps militaris mycelium material prepared by the present invention with an exogenous carboxyl group-containing monomer having antibacterial properties.

[0036] Suitable monomers containing carboxyl groups may be antimicrobial peptides, carboxypenicillins, and β-lactam antibiotics except monocyclic β-lactams.

[0037] The following is a detailed description of the Cordyceps militaris mycelium material and its preparation method and application described in the embodiments of the present invention in conjunction with specific examples.

[0038] Example 1 (1) Preparation of Cordyceps militaris spore suspension: 20 μL of the Cordyceps militaris strain was taken from -80°C and activated on a potato solid culture medium, and then placed in a 28°C incubator for 7 days to obtain a first-generation Cordyceps militaris strain. The aerial mycelium on the culture medium was then washed with an appropriate amount of pure water and then transferred to an eggplant bottle, which was then placed in a 28°C incubator for 7 days to obtain a second-generation Cordyceps militaris strain. The eggplant bottle culture medium was washed with 50 mL of pure water and then filtered through sterile gauze to obtain a Cordyceps militaris spore suspension.

[0039] The preparation method of the above-mentioned potato solid culture medium is as follows: weigh 200g of peeled potatoes, chop them, put them into pure water and boil them for 30 minutes, filter the potato residue with double-layer gauze to obtain the filtrate, add 20g of glucose and 15g of agar powder, make the volume to 1000mL, no need to adjust the pH, stir evenly and pour into a blue-capped bottle, put it into a sterilizer at 115°C, and sterilize it for 30 minutes.

[0040] (2) Liquid culture of Cordyceps militaris: Take 5 μL of Cordyceps militaris spore suspension and inoculate it into 100 mL of potato glucose water and place it in a shaker for liquid culture for 5 days. The pH of the liquid culture medium and the culture environment temperature are regulated by single factors, and yeast extract and glycerol are added (see Table 1 for the specific regulation scheme). After the spores germinate, the Cordyceps militaris ball culture liquid with uniform winding size is obtained.

[0041] Table 1: Liquid culture medium single factor regulation scheme (3) Preparation of Cordyceps militaris mycelium material: The culture solution of Cordyceps militaris obtained by culturing under the culture conditions (culture medium pH=7, culture temperature 28°C, glycerol addition amount 6%, yeast extract addition amount 8%) in the above step (2) was filtered and repeatedly washed three times to obtain bacterial balls, and the filtered bacterial balls were placed in a culture dish and dried for 5 hours to obtain Cordyceps militaris mycelium material.

[0042] (4) Chitinase treatment of Cordyceps militaris mycelium: Take 1 g of the Cordyceps militaris mycelium material prepared in step (3) above, add 1 mL of a 0.5 g / mL chitinase solution, set the temperature at 28° C., and perform enzymolysis for 1 h to obtain the Cordyceps militaris mycelium material.

[0043] (5) Antimicrobial peptide grafting reaction: Dissolve the antimicrobial peptide in MES (2-morpholineethanesulfonic acid) buffer at a concentration of 5 mg / mL. Ensure that the pH of the solution is 5.5-6.0. Add EDC solution to the antimicrobial peptide solution to a final concentration of 0.1 M. Immediately add NHS solution to a final concentration of 0.05 M. Stir at room temperature for 20 minutes to activate the carboxyl group in the antimicrobial peptide to form NHS ester for coupling reaction. Then add 1 mL of the activated antimicrobial peptide solution dropwise to the Cordyceps militaris mycelium material obtained in step (4) above. Stir the reaction at room temperature for 4 hours. Adjust the pH of the solution to 7.0 to terminate the reaction and obtain the Cordyceps militaris mycelium antimicrobial material.

[0044] 1. Effects of different culture conditions on the content of β-glucan in Cordyceps militaris The Cordyceps militaris ball culture liquid obtained by single-factor regulation of the culture conditions in step (2) of Example 1 was filtered through a double-layer gauze and washed several times to remove the residual culture medium, placed in a 60° oven for 24 hours for drying, ground into powder with a mortar, and then passed through an 80-mesh sieve to prepare Cordyceps militaris mycelium powder. Take 0.1 g of Cordyceps militaris mycelium powder, take 1 mL of a mixed solution of chloroform and n-butanol pre-prepared in a volume ratio of 4:1, mix the two, shake thoroughly for 30 minutes, and then centrifuge for 1 minute, then use a pipette to separate the aqueous phase from the chloroform phase, and repeat this operation 3 times. After fully removing the impurities, add 2 mL of pure water to the sample and use a cell disruptor to break the cell wall for 30 minutes. Then add 0.2 mL of 6M NaOH solution and incubate in an 80°C water bath for 30 minutes. The purpose of this process is to dissolve the glucan in the cell wall. Then add 2.1 mL of the pre-prepared 0.03% aniline blue solution to each sample. Aniline blue can specifically bind to the 1,3 glycosidic bond of β-glucan. Figure 1 As shown. The sample was vortexed briefly and incubated in a 50°C water bath for 30 minutes, and then placed in a 20°C water bath for 30 minutes to obtain a stained solution. Five parallel samples were prepared for each sample and added to a ninety-six-well plate. Under the conditions of an excitation wavelength of 400nm and an emission wavelength of 460nm, the fluorescence was quantitatively measured using an enzyme reader. The fluorescence intensity was used to determine whether the β-glucan content was increased compared to the control group. Figure 3 Figure 4 Figure 5 Figure 6 The results show that the intensity of fluorescence under different culture conditions reflects the content of β-glucan. The results show that the optimal culture conditions are: culture temperature 28℃, pH=7, yeast extract addition amount 8%, glycerol addition amount 6%.

[0045] The preparation method of the above 0.03% aniline blue solution is as follows: weigh 0.15 g of aniline blue powder, dilute to 500 mL with ethanol solution, and store in a dark place.

[0046] 2. Determination of β-glucan content in Cordyceps militaris mycelium material before and after chitinase hydrolysis The culture temperature in step (2) of Example 1 was 28°C, pH=7, the amount of yeast extract added was 8%, and the amount of glycerol added was 6%. The Cordyceps militaris ball culture solution obtained in the experimental group was filtered with double-layer gauze and washed several times to remove the residual culture medium, placed in a 60°C oven for 24 hours for drying, and then ground into powder with a mortar and sieve through an 80-mesh sieve to prepare Cordyceps militaris mycelium powder. 0.1g of the Cordyceps militaris mycelium powder was added with 1mL of chitinase solution, and placed in a 28°C water bath for 1 hour, during which the shaking was repeated 3 times to fully enzymatically hydrolyze the chitin. In addition, 0.1g of Cordyceps militaris mycelium powder was taken and 1mL of deionized water was added as the control group. All samples were prepared in triplicate. Then 2.1mL of aniline blue solution was taken and added to each sample. The sample was briefly vortexed and incubated in a 50°C water bath for 30 minutes, and then placed in a 20°C water bath for 30 minutes to obtain a dyed solution. Under the conditions of excitation wavelength of 400nm and emission wavelength of 460nm, fluorescence was quantitatively measured using an enzyme-labeled instrument. Figure 2 As shown, the fluorescence intensity of Cordyceps militaris mycelium treated with chitinase was significantly higher than that of Cordyceps militaris mycelium without enzyme treatment.

[0047] Preparation of the above aniline blue solution: weigh 3.285 mL of HCL solution and 18.39 g of glycine, dilute to 500 mL with 0.03% aniline blue solution, and adjust the pH to 9.5 with 6 mol NaOH solution for later use.

[0048] 3 Antibacterial effect determination Take the above-mentioned mycelium antibacterial material before chitin enzymatic hydrolysis and the Cordyceps militaris mycelium antibacterial material after chitin enzymatic hydrolysis obtained in step (5) respectively. Add 0.2 mL of Escherichia coli solution to the surface of the material in a clean bench, incubate for 0.5-1 h, then wash the bacteria off the material with PBS solution, then take 0.2 mL of the washed PBS solution to spread on the plate, incubate at 37°C for 6-8 h, then check their survival and activity on the agar plate by the typical single colony method, calculate the number of single colonies, and prove the antibacterial performance.

[0049] like Figure 7-8 As shown in the figure, the results prove that the mycelium antibacterial material after chitin enzymatic hydrolysis has an increased β-glucan content, and the glucan surface contains rich hydroxyl groups, which is easy to be chemically modified. Therefore, the material is grafted with more antimicrobial peptide components. Calculated by the number of colonies, its antibacterial performance is improved by 420% compared with the antibacterial mycelium material prepared without chitinase hydrolysis.

Claims

1. A method for preparing Cordyceps militaris mycelium material, characterized in that: The following steps are involved: (1) inoculating a Cordyceps militaris spore suspension into a potato glucose water culture medium containing yeast extract and glycerol for culturing, filtering and washing to obtain a bacterial ball, and placing the bacterial ball in a container and drying it to obtain a Cordyceps militaris mycelium material precursor; (2) Adding a chitinase solution to the Cordyceps militaris mycelium material precursor of step (1) for enzymatic hydrolysis to obtain a Cordyceps militaris mycelium material.

2. The preparation method according to claim 1, characterized in that: The culture temperature in step (1) is 24°C to 30°C, the pH is 5 to 8, and the culture time is 60 to 80 hours.

3. The preparation method according to claim 1, characterized in that: The drying time in step (1) is 4 to 6 hours.

4. The preparation method according to claim 1, characterized in that: In the potato glucose water culture medium of step (1), the amount of yeast extract added is 2% to 8%, and the amount of glycerol added is 4% to 8%.

5. The preparation method according to claim 1, characterized in that: The concentration of the chitinase solution in step (2) is 0.4-0.6 g / mL, and the added amount is 1 mL per 1 g of Cordyceps militaris mycelium material.

6. The preparation method according to claim 1, characterized in that: The enzymatic hydrolysis temperature in step (2) is 26-30° C. and the enzymatic hydrolysis time is 0.5-1 h.

7. The preparation method according to claim 1, characterized in that: Also includes the steps, (3) Grafting a monomer having antibacterial function onto the Cordyceps militaris mycelium material described in step (2).

8. A Cordyceps militaris mycelium material, characterized in that: Prepared according to the preparation method according to any one of claims 1 to 7.

9. An application of Cordyceps militaris mycelium material in preparing antibacterial mycelium material, characterized in that: The Cordyceps militaris mycelium prepared by the preparation method of claim 1 is subjected to a grafting reaction with a monomer having an antibacterial function to obtain an antibacterial mycelium material.

10. The use according to claim 9, characterized in that: The monomer with antibacterial function is an antibacterial peptide.

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