Method for efficiently extracting melanin from melanospora oryzae hyphae

Abstract

The invention is applicable to the technical field of melanin extraction, and provides a method for efficiently extracting melanin from melanospora oryzae hyphae, which comprises the following steps: activating melanospora oryzae until the activity is recovered; punching the rice nightshade fungus cake of which the activity is recovered, and then inoculating the rice nightshade fungus cake into a PDB culture medium for culturing; performing centrifugal treatment on the fermented liquid obtained after culture to obtain hyphae, and then drying the hyphae; grinding the dried hyphae, placing the ground hyphae in a NaOH solution for boiling water bath, and extracting melanin; carrying out centrifugal treatment on the NaOH solution dissolved with the melanin, taking supernate after centrifugation, adjusting the pH value, and standing to precipitate the melanin; and centrifuging the treated supernatant, discarding the supernatant, taking the precipitate, and drying to obtain the melanin. According to the method, the variety of the culture medium is changed, a solid culture medium is replaced by a liquid culture medium, and the melanin in the nightshade is extracted by culturing the fermentation liquor, so that the extraction efficiency and the extraction quality of the melanin of fungal hyphae are improved, the operation is simple, and the cost is relatively low.

Description

Technical Field

[0001] The invention belongs to the technical field of melanin extraction, and in particular relates to a method for efficiently extracting melanin from rice black spore hyphae. Background Art

[0002] The colony of Nigrospora oryzae is white or gray in the early stage of growth, and black or gray-black in the later stage. A large amount of melanin will accumulate in the later stage of growth. The melanin in Nigrospora oryzae is closely related to its pathogenicity. As a macromolecular polymer, fungal melanin has a wide range of biological functions, which helps the fungi resist environmental factors that are not conducive to their own growth, thereby improving the survival ability of the fungi. In addition, melanin can also be used as an antioxidant, ultraviolet absorber, antiviral drug, free radical scavenger, photoprotectant of biological pesticides, food pigments and chemical dyes, etc. It has been widely used in many fields such as pharmaceutical chemicals, food industry, cosmetics industry, and agriculture.

[0003] However, current methods for extracting fungal melanin extract low levels of melanin. Summary of the invention

[0004] The purpose of the embodiments of the present invention is to provide a method for efficiently extracting melanin from the mycelium of Nigrosporium oryzae, aiming to solve the problems existing in the above-mentioned background technology.

[0005] The embodiment of the present invention is achieved by a method for efficiently extracting melanin from black spore mycelium of rice, comprising the following steps: (1) activating the rice black spore fungus to restore its activity; (2) punching holes in the recovered rice black spore fungus cake and inoculating it into PDB medium for cultivation; (3) centrifuging the fermentation liquid obtained after the culture to obtain mycelium, and then drying it; (4) Grind the dried mycelium and place it in a boiling water bath with a NaOH solution to extract melanin; (5) Centrifuging the NaOH solution containing melanin, taking the supernatant, adjusting the pH value, and standing to precipitate the melanin; (6) Centrifuge the treated supernatant and discard the supernatant, take the precipitate and dry it to obtain melanin.

[0006] Preferably, the step of activating the rice black spore to restore activity is specifically: transferring the rice black spore on the PDA slope stored in a 4°C refrigerator to a PDA culture dish, and after it grows for 1 to 2 days, cutting the edge hyphae into pieces and transferring them to a new PDA culture dish again, and transferring them continuously for 2 to 3 times to activate the rice black spore to restore activity.

[0007] Preferably, the step of punching the restored activity of the rice black spore fungus cake and inoculating it into the PDB medium for cultivation is specifically: punching the restored activity of the rice black spore fungus with a hole puncher, inoculating it into 100-200 mL of PDB medium, placing it in a shaker, setting the temperature to 25-28° C. and the speed to 180-200 r / min for dark cultivation.

[0008] Preferably, the step of centrifuging the fermentation liquid obtained after the culture to obtain mycelium is specifically: centrifuging the fermentation liquid after dark culture at 3900r / min for 15-20min to collect the mycelium.

[0009] Preferably, the step of grinding the dried mycelium and placing it in a NaOH solution in a boiling water bath is specifically: grinding the dried mycelium and placing it in a 30 mL 1 mol / L NaOH solution in a boiling water bath for 4 to 5 hours, shaking it every 30 minutes, and replenishing the NaOH solution in time.

[0010] Preferably, the step of centrifuging the NaOH solution containing melanin, taking the supernatant after centrifugation, adjusting the pH value, and standing to precipitate the melanin is specifically as follows: centrifuging the NaOH solution containing melanin at 3900r / min for 15-20min to take the supernatant, adjusting the pH value of the supernatant to between 2.0 and 3.0 with 1mol / L NaOH and 7mol / L HCl, and then placing it in a 4°C refrigerator for 2-3d to precipitate the melanin.

[0011] Preferably, the step of centrifuging the treated supernatant and discarding the supernatant, and drying the precipitate is specifically as follows: centrifuging the supernatant after static precipitation at 3900r / min for 15-20min and discarding the supernatant, and drying the obtained precipitate in an oven at 60-70°C.

[0012] The embodiment of the present invention provides a method for efficiently extracting melanin from rice black spore hyphae, which changes the type of culture medium, replaces the solid culture medium with a liquid culture medium, and cultivates the fermentation liquid to extract the melanin in the rice black spore, thereby improving the extraction efficiency and extraction quality of the fungal hyphae melanin, is simple to operate, and has low cost. BRIEF DESCRIPTION OF THE DRAWINGS

[0013] Figure 1 Schematic diagram of the fermentation liquid of Nigrosporium oryzae at three stages provided in Example 1 of the present invention; Figure 2 Schematic diagram of the phenotypes of Mycelium oryzae after drying and grinding at three stages provided in Example 1 of the present invention; Figure 3 Schematic diagram of NaOH solution containing melanin from Nigrospora oryzae at three stages provided in Example 1 of the present invention; Figure 4 Schematic diagram of the melanin of Nigrospora oryzae at three stages provided in Example 1 of the present invention. DETAILED DESCRIPTION

[0014] In order to make the purpose, technical solution and advantages of the present invention more clearly understood, the present invention is further described in detail below in conjunction with the accompanying drawings and embodiments. It should be understood that the specific embodiments described herein are only used to explain the present invention and are not intended to limit the present invention.

[0015] A method for efficiently extracting melanin from rice black spore hyphae comprises the following steps: (1) Transfer the rice black spores on the PDA slope stored in a 4°C refrigerator to a PDA culture dish. After it grows for 1-2 days, cut the edge hyphae into pieces and transfer them to a new PDA culture dish. Repeat the transfer 2-3 times to activate the rice black spores until they are active again. (2) Punch the rice black spore fungus obtained in step (1) with a hole puncher of 8 mm diameter, inoculate it into 100-200 mL PDB culture medium, place it in a shaker, set the temperature to 25-28°C and the speed to 180-200 r / min for dark culture; (3) Pour the fermentation liquid obtained in step (2) into a 50 mL centrifuge tube, centrifuge at 3900 r / min for 15-20 min to collect the mycelium, and then place it in a 70°C oven to dry; (4) Grind the mycelium obtained in step (3) and place it in 30 mL of 1 mol / L NaOH solution in a boiling water bath for 4 to 5 hours to extract melanin, shaking it every 30 minutes and replenishing the NaOH solution in time. After the boiling water bath, the melanin has been fully dissolved in the NaOH solution; (5) Centrifuge the mixture obtained in step (4) at 3900 r / min for 15-20 min to obtain the supernatant, adjust the pH value of the supernatant to between 2.0 and 3.0 with 1 mol / L NaOH and 7 mol / L HCl, and place in a refrigerator at 4°C for 2-3 days to precipitate melanin; (6) The mixture obtained in step (5) is centrifuged at 3900 r / min for 15-20 min, and the supernatant is discarded. The obtained precipitate is placed in an oven at 60-70°C and dried to obtain melanin.

[0016] The specific implementation of the present invention is described in detail below in conjunction with specific embodiments.

[0017] The Nigrospora oryzae strain used in the examples was isolated from diseased leaves of Euonymus ilex collected from the East District of Nanyang Normal University; The PDB medium in the embodiment needs to be sterilized at 115°C for 30 minutes after preparation and stored at room temperature; In the examples, 1 mol / L NaOH and 7 mol / L HCl solutions were prepared before use and stored at room temperature. Example 1

[0018] A method for efficiently extracting melanin from rice black spore hyphae comprises the following steps: (1) Transfer the rice black spores on the PDA slope stored in a 4°C refrigerator to a PDA culture dish. After it grows for 1-2 days, cut the edge hyphae into pieces and transfer them to a new PDA culture dish. Repeat the transfer 2-3 times to activate the rice black spores until they are active again. (2) The rice black spore fungus obtained in step (1) was punched with an 8 mm diameter puncher and inoculated into 100 mL of PDB medium. The medium was placed in a shaker and the temperature was set to 25°C and the speed was set to 180 r / min for dark culture. The culture was divided into three periods: early stage (2 days), middle stage (4 days), and late stage (7 days). Figure 1 As shown; (3) Pour the fermentation liquid obtained in step (2) into a 50 mL centrifuge tube, centrifuge at 3900 r / min for 20 min to collect the mycelium, and then dry it in an oven at 70°C. Figure 2 As shown; (4) Grind the mycelium obtained in step (3) and place it in 30 mL of 1 mol / L NaOH solution in a boiling water bath for 5 h to extract melanin. During this period, shake it every 30 min and replenish the NaOH solution in time. After the boiling water bath, the melanin has been fully dissolved in the NaOH solution. Figure 3 As shown; (5) Centrifuge the mixture obtained in step (4) at 3900 r / min for 20 min to obtain the supernatant, adjust the pH value of the supernatant to between 2.0 and 3.0 with 1 mol / L NaOH and 7 mol / L HCl, and place in a refrigerator at 4°C for 3 days to precipitate melanin; (6) The mixture obtained in step (5) is centrifuged at 3900 r / min for 20 min, the supernatant is discarded, and the obtained precipitate is dried in an oven at 70°C to obtain melanin. Figure 4 shown.

[0019] According to the test, 100 mL of fermentation liquid contained 41.17 mg / g of DHN melanin in the body of the black spore fungus in the late growth stage. Streptomyces kathirae ) The optimal culture composition tried produced 13.6 g / L of melanin, so the method of the embodiment of the present invention significantly improved the extraction efficiency and extraction quality. Example 2

[0020] A method for efficiently extracting melanin from rice black spore hyphae comprises the following steps: (1) Transfer the rice black spores on the PDA slope stored in a 4°C refrigerator to a PDA culture dish. After it grows for 1-2 days, cut the edge hyphae into pieces and transfer them to a new PDA culture dish. Repeat the transfer 2-3 times to activate the rice black spores until they are active again. (2) Punch the black spore fungus obtained in step (1) with an 8 mm diameter puncher and inoculate it into 150 mL of PDB medium, place it in a shaker, set the temperature to 26°C and the speed to 190 r / min for dark culture; (3) Pour the fermentation liquid obtained in step (2) into a 50 mL centrifuge tube, centrifuge at 3900 r / min for 15 min to collect the mycelium, and then place it in a 70°C oven to dry; (4) Grind the mycelium obtained in step (3) and place it in 30 mL of 1 mol / L NaOH solution in a boiling water bath for 4 h to extract melanin, shaking it every 30 min and replenishing the NaOH solution in time. After the boiling water bath, the melanin has been fully dissolved in the NaOH solution; (5) Centrifuge the mixture obtained in step (4) at 3900 r / min for 15 min to obtain the supernatant, adjust the pH value of the supernatant to between 2.0 and 3.0 with 1 mol / L NaOH and 7 mol / L HCl, and place in a refrigerator at 4°C for 2 days to precipitate melanin; (6) The mixture obtained in step (5) was centrifuged at 3900 r / min for 15 min, and the supernatant was discarded. The obtained precipitate was placed in an oven at 60°C and dried to obtain melanin. Example 3

[0021] A method for efficiently extracting melanin from rice black spore hyphae comprises the following steps: (1) Transfer the rice black spores on the PDA slope stored in a 4°C refrigerator to a PDA culture dish. After it grows for 1-2 days, cut the edge hyphae into pieces and transfer them to a new PDA culture dish. Repeat the transfer 2-3 times to activate the rice black spores until they are active again. (2) Punch the rice black spore fungus obtained in step (1) with an 8 mm diameter puncher and inoculate it into 200 mL of PDB medium, place it in a shaker, set the temperature to 28° C. and the speed to 200 r / min for dark culture; (3) Pour the fermentation liquid obtained in step (2) into a 50 mL centrifuge tube, centrifuge at 3900 r / min for 18 min to collect the mycelium, and then place it in a 70°C oven to dry; (4) Grind the mycelium obtained in step (3) and place it in 30 mL of 1 mol / L NaOH solution in a boiling water bath for 4.5 h to extract melanin. During this period, shake it once every 30 min and replenish the NaOH solution in time. After the boiling water bath, the melanin has been fully dissolved in the NaOH solution; (5) Centrifuge the mixture obtained in step (4) at 3900 r / min for 18 min to obtain the supernatant, adjust the pH value of the supernatant to between 2.0 and 3.0 with 1 mol / L NaOH and 7 mol / L HCl, and place in a refrigerator at 4°C for 2 days to precipitate melanin; (6) The mixture obtained in step (5) was centrifuged at 3900 r / min for 18 min, and the supernatant was discarded. The obtained precipitate was placed in an oven at 65°C and dried to obtain melanin.

[0022] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions and improvements made within the spirit and principles of the present invention should be included in the protection scope of the present invention.

Claims

1. A method for efficiently extracting melanin from black spore mycelium of rice, It is characterized in that The following steps are involved: (1) activating the rice black spore fungus to restore its activity; (2) punching holes in the recovered rice black spore fungus cake and inoculating it into PDB medium for cultivation; (3) centrifuging the fermentation liquid obtained after the culture to obtain mycelium, and then drying it; (4) Grind the dried mycelium and place it in a boiling water bath with a NaOH solution to extract melanin; (5) Centrifuging the NaOH solution containing melanin, taking the supernatant, adjusting the pH value, and standing to precipitate the melanin; (6) Centrifuge the treated supernatant and discard the supernatant, take the precipitate and dry it to obtain melanin.

2. The method for efficiently extracting melanin from black spore mycelium of rice according to claim 1, It is characterized in that The step of activating the rice black spore to restore activity is specifically: transferring the rice black spore on the PDA slope stored in a 4°C refrigerator to a PDA culture dish, growing for 1 to 2 days, and then transferring the edge hyphae to a new PDA culture dish again, and the transfer is repeated 2 to 3 times to activate the rice black spore to restore activity.

3. The method for efficiently extracting melanin from black spore mycelium of rice according to claim 1, It is characterized in that The step of punching the recovered black spore fungus cake and then inoculating it into the PDB culture medium for cultivation is specifically: punching the recovered black spore fungus with a hole puncher, inoculating it into 100-200 mL of PDB culture medium, putting it into a shaking table, setting the temperature to 25-28° C. and the rotation speed to 180-200 r / min for dark cultivation.

4. The method for efficiently extracting melanin from black spore mycelium of rice according to claim 1, It is characterized in that The step of centrifuging the fermentation liquid obtained after the culture to obtain mycelium is specifically: centrifuging the fermentation liquid after dark culture at 3900r / min for 15-20min to collect the mycelium.

5. The method for efficiently extracting melanin from the mycelium of Nigrospora oryzae according to claim 1, It is characterized in that The step of grinding the dried mycelium and placing it in a boiling water bath with a NaOH solution is specifically as follows: grinding the dried mycelium and placing it in a boiling water bath with a 30 mL 1 mol / L NaOH solution for 4 to 5 hours, shaking it every 30 minutes, and replenishing the NaOH solution in time.

6. The method for efficiently extracting melanin from the mycelium of Nigrospora oryzae according to claim 1, It is characterized in that The step of centrifuging the NaOH solution containing melanin, taking the supernatant after centrifugation, adjusting the pH value, and standing to precipitate the melanin specifically comprises: centrifuging the NaOH solution containing melanin at 3900 r / min for 15-20 min to take the supernatant, adjusting the pH value of the supernatant to between 2.0 and 3.0 with 1 mol / L NaOH and 7 mol / L HCl, and then placing the supernatant in a 4°C refrigerator for 2-3 days to precipitate the melanin.

7. The method for efficiently extracting melanin from the mycelium of Nigrospora oryzae according to claim 1, It is characterized in that The step of centrifuging the treated supernatant and discarding the supernatant, and drying the precipitate is specifically as follows: centrifuging the supernatant after static precipitation at 3900r / min for 15-20min and discarding the supernatant, and drying the obtained precipitate in an oven at 60-70°C.

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