TWI509071B - Method for inducing secondary metabolites production of fungus utilizing apoptosis mechanism - Google Patents

Description

Method for inducing biosynthesis of secondary metabolites of fungi

The invention relates to a method for inducing biosynthesis of secondary metabolites of fungi, and in particular to a method for inducing biosynthesis of secondary metabolites of fungi by utilizing a biochemical mechanism of apoptosis.

In recent years, the medical value of fungi has been increasingly valued. For example, Chinese herbal medicines or fungi used to produce antibiotics have high medical value. In the case of Chinese herbal fungi, it has been tried as a traditional Chinese medicine in the Republic of China, and its official record has been for decades. The fungi used in Chinese herbal medicines include Ganoderma lucidum, burdock and Brazilian mushrooms, among which Astragalus and Ganoderma lucidum are the largest. The above-mentioned main medical value of the fungus, whether applied to Chinese herbal medicine or used to produce antibiotics, is its secondary (secondary) metabolites, such as triterpenoids, ricinic acid, cordycepin, lovastatin ( Lovastatin), penicillin, etc.; in which Ganoderma lucidum is used as an example, it belongs to the basidiomycete fungus, and it has been widely used in folk medicine or health foods such as Chinese herbal medicine for thousands of years. Ganoderma lucidum can be used to assist in the medical treatment of diseases such as cancer, hepatitis, hypertension and heart disease. In addition, Ganoderma Lucidum can also be used to enhance immunity. And used to improve physical fitness. Among them, the above-mentioned effects of Ganoderma lucidum mainly come from the most precious secondary metabolites of Ganoderma lucidum containing triterpenoids, and the main component of triterpenoids includes Ganoderic acids. In the past, it has been pointed out that the ganoderic acid extracted and extracted from Ganoderma lucidum has many biological activities such as anticancer activity, antiviral activity, liver protective activity, antioxidant activity, and inhibition of histamine release. In addition, recent studies have also found that Ganoderma lucidum T can inhibit the metastasis of cancer cells by inhibiting the activation of NF-κB.

However, the yield and yield of secondary metabolites extracted from fungi are extremely low, thus making secondary metabolites with biological activity and medical value, such as the aforementioned triterpenoids, including ganoderic acid, etc., more precious, resulting in The price is high. Accordingly, how to increase the yield and yield of secondary metabolites in fungal mycelium has been a very important topic in this technical field; if it can mass produce secondary metabolites with high medical value, it can make more People who need these natural ingredients are benefited.

In view of the above, the present invention provides a method capable of increasing the yield and yield of secondary metabolites of fungi, which can increase the yield of secondary metabolites of fungi by two to three times compared with the prior art.

One embodiment of the present invention provides a method for inducing biosynthesis of secondary metabolites of fungi by forcing fungal cells to induce apoptosis and inducing secondary synthesis of secondary metabolites, and inducing secondary metabolism of fungi. The method for biosynthesis of a product comprises providing a mycelium of a fungus, and cultivating the mycelium for a predetermined incubation time, and having an effective concentration of one The apoptosis inducer is transported to the mycelium and cultured for a predetermined induction time. Finally, the secondary metabolite of the mycelium is extracted and purified to obtain a secondary metabolite comprising Gandoric acid. .

According to one embodiment of the present invention, the method for inducing biosynthesis of secondary metabolites of Ganoderma lucidum, wherein the fungus may be a fungus such as Antrodia camphorata, Cordyceps sinensis, Brazilian mushroom, Yunzhi, Ganoderma lucidum.

According to one embodiment of the present invention, the method for inducing biosynthesis of secondary metabolites of fungi, wherein the predetermined culture time is from 4 days to 18 days.

According to one embodiment of the present invention, the method for inducing biosynthesis of secondary metabolites of the fungus, wherein the Ganoderma lucidum is deposited in the Bioresource Conservation and Research Center of the Food Industry Development Research Institute, has the registration number BCRC 36111.

According to one embodiment of the present invention, the method for inducing biosynthesis of a secondary metabolite of a fungus, wherein the apoptosis inducer is aspirin, and the effective concentration is 0.5 millimolar (mM) ) to a concentration of 8 millimolar (mM), and the preset induction time is 6 hours to 48 hours.

According to one embodiment of the present invention, the method for inducing biosynthesis of a secondary metabolite of a fungus, wherein the inducer of apoptosis is 1-chloro-2,4-dinitrobenzene (1-Chloro-2, 4) -dinitrobenzene), and its effective concentration is from 0.1 millimolar (mM) to 1 millimolar (mM), and the preset induction time is from 1 day to 12 days.

According to one embodiment of the present invention, the method for inducing biosynthesis of a secondary metabolite of a fungus, wherein the inducer of apoptosis is hydrogen peroxide (H ₂ O ₂ ), and the effective concentration thereof is 0.8 millimolar. (mM) to 2.4 millimolar (mM) and the preset induction time is from 1 day to 12 days.

The technology and aspects of the present invention will be described in the following, and the present invention will be apparent to those of ordinary skill in the art.

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The above and other objects, features, advantages and embodiments of the present invention will become more <RTIgt; </ RTI> <RTIgt; </ RTI> <RTIgt; Comparison of the yields of different effective concentrations of apoptosis inducer (aspirin) on the total amount of ganoderic acid contained in the mycelium of Ganoderma lucidum; Figure 3 shows the different induction of apoptosis inducer (aspirin) Comparison of the yield of total ginseng acid in Ganoderma lucidum mycelium in time; Figure 4 shows the different culture time of Ganoderma lucidum mycelium (starting inoculum) before the addition of apoptosis inducer for induction of Ganoderma lucidum mycelium Comparison of the yield of the total amount of ganoderic acid contained in the body; Figure 5 is a comparison chart of the effect of the inducer of apoptosis (hydrogen peroxide) on the total yield of ganoderic acid contained in the mycelium of Ganoderma lucidum; Comparison of the effect of apoptosis inducer (1-chloro-2,4-dinitrobenzene) on the yield of total ginseng acid in Ganoderma lucidum mycelium; and Figure 7 is the use of DNA break end labeling test (TUNEL) Assay photomicrograph of apoptosis inducer (aspirin) on apoptosis status of ganoderma lucidum cells .

Embodiments of the invention are discussed in more detail below. However, this embodiment can be applied to various inventive concepts and can be embodied in various specific ranges. The specific embodiments are for illustrative purposes only and are not limited by the scope of the disclosure.

The technology and aspects of the present invention will be described in the following, and the present invention will be apparent to those of ordinary skill in the art.

Please refer to FIG. 1 , which is a flowchart of a method according to an embodiment of the present invention. The invention provides a method for inducing biosynthesis of secondary metabolites of fungi, which is a method for forcing fungal cells to induce apoptosis and inducing biosynthesis of secondary metabolites, and the method for inducing biosynthesis of secondary metabolites of fungi comprises the following steps: Step 100: Providing a mycelium of a fungus, and culturing the mycelium for a predetermined incubation time, wherein the preset culture time may be 4 days to 18 days. Step 200: Transfer an apoptosis inducer having an effective concentration to the mycelium, and culture it for a predetermined induction time, wherein the effective concentration and the induction time are different depending on the apoptosis inducer used. Difference; Step 300: Extract and purify the secondary metabolite of the mycelium to obtain the secondary metabolite.

In addition, the aforementioned fungi may be fungi such as Antrodia camphorata, Cordyceps sinensis, Brazilian mushroom, Yunzhi, Ganoderma lucidum; the following test examples are exemplified by only Ganoderma lucidum, which is not intended to limit the scope of the claimed invention.

The invention is further illustrated by the following specific test examples. The following test examples are provided by way of illustration only and are not intended to limit the scope of the invention.

[Test example]

The Ganoderma lucidum strain of this test was purchased from the Center for Biological Resource Conservation and Research of the Food Industry Development Research Institute under the registration number BCRC 36111.

The Ganoderma lucidum strain was inoculated into a petri dish of potato dextrose agar (PDA) and cultured in an environment of 28 °C. After 7 to 10 days of culture, the portion of the mycelium was taken as the starting inoculum for the subsequent test.

In order to test the effect of the apoptosis inducer on the secondary metabolites of Ganoderma lucidum, the aforementioned initial inoculum is placed in the sterilized water, and then uniformly dispersed by a sterilized stirring rod; wherein the ratio of the initial inoculum to the sterilized water is added. About 8.75g: 50ml, 70mg of the starting inoculum (Ganoderma lucidum mycelium) was extracted and inoculated into a PDA culture dish (9 cm diameter culture dish), and cultured in an environment of 28 ° C. 1 to 12 days. After that, the Ganoderma lucidum mycelium was transferred to a 250 ml Erlenmeyer flask containing 25 ml of potato dextrose broth, and the apoptosis inducer was added and mixed uniformly and placed on a 100 rpm incubator for 6 hours to 4 days to force the Ganoderma lucidum. The mycelium begins to undergo apoptosis to induce and enhance the total yield of the ginseng acid contained therein. The "*" in the pattern of this test example represents a statistical difference (p value), where "*" represents p<0.05, "**" represents p<0.01, and "***" represents p<0.001. .

First, the inducer of apoptosis is the induction condition test of aspirin

Please refer to Fig. 2, which is a graph comparing the yields of total ginseng acids contained in the mycelium of Ganoderma lucidum with different effective concentrations of apoptosis inducer (aspirin). Ganoderma lucidum mycelium (starting inoculum) was cultured in a temperature environment of 28 ° C for 4 days, and then mixed with 0.5 mM, 1 mM, 2 mM, 4 mM and 8 mM aspirin for 1 day to force Ganoderma lucidum The filaments begin to undergo apoptosis to induce and enhance the total yield of ganoderic acid contained therein. As shown, an increase in the concentration of aspirin promotes the amount (yield) of the performance of the Garodic acid. Among them, the concentration of aspirin at 4 mM and 8 mM was preferred. Therefore, 4 mM aspirin was selected to evaluate the effect of the different induction time on the yield of the total amount of ganoderic acid contained in the ganoderma lucidum mycelium.

Please refer to Fig. 3 together, which is a comparison chart showing the different induction time of apoptosis inducer (aspirin) on the total amount of ginseng acid contained in the mycelium of Ganoderma lucidum. As shown in the figure, when 4 mM apoptosis inducer (aspirin) was mixed and cultured at 28 ° C for 4 days in Ganoderma lucidum mycelium (starting inoculum), it was cultured (induced) for 6 hours and 12 hours, respectively. At 24 hours and 48 hours, the amount of Ganoderic acid (yield) was measured. The yield of total amount of ganoderic acid increased significantly from the sixth hour after the addition of aspirin. The yield of the total amount of Ganoderma lucidum from the 12th hour was preferred; therefore, 4 mM aspirin was selected and the induction time of 24 hours was used to evaluate the temperature environment of different Ganoderma lucidum mycelium (starting inoculum) at 28 ° C. Cultivation Whether the time affects the yield of the total amount of ganoderic acid.

Please refer to Fig. 4, which compares the yields of total ginseng acid contained in Ganoderma lucidum mycelium after induction of Ganoderma lucidum mycelium (initial inoculum) before the addition of apoptosis inducer. Figure. When 4 mM apoptosis inducer (aspirin) was mixed at 28 ° C for 4 days, 8 days and 12 days of Ganoderma lucidum mycelium (starting inoculum), it was induced for 24 hours and measured. The amount of Ganoderma acid (yield); the fourth day after the addition of aspirin, the total yield of Ganoderma lucidum increased significantly, and the total amount of Ganoderma lucidum from Day 12 The yield of the amount is preferred.

Second, the inducer of apoptosis is the induction condition test of hydrogen peroxide

Please refer to Fig. 5, which is a comparison chart showing the effect of the apoptosis inducer (hydrogen peroxide) on the yield of the total amount of ganoderic acid contained in the mycelium of Ganoderma lucidum. Ganoderma lucidum mycelium (starting inoculum) was cultured in a temperature environment of 28 ° C for 4 days, and then cultured (induced) with 1.6 mM hydrogen peroxide for 4 days to force the mycelium of Ganoderma lucidum to start cell withering. The effect of death, in order to induce and enhance the total yield of ganoderic acid contained in it. Among them, the control group is a ganoderma mycelium that does not add hydrogen peroxide. As shown in the figure, the addition of hydrogen peroxide does promote the amount of expression (yield) of ganoderic acid.

3. Induction condition test for apoptosis inducer 1-chloro-2,4-dinitrobenzene

Please refer to Fig. 6, which is a comparison chart showing the effect of the apoptosis inducer (1-chloro-2,4-dinitrobenzene) on the yield of the total amount of ganoderic acid contained in the mycelium of Ganoderma lucidum. Ganoderma lucidum mycelium (starting inoculum) was cultured in a temperature environment of 28 ° C for 4 days, and then mixed with 0.4 mM 1-chloro-2,4-dinitrobenzene for 4 days. Ganoderma lucidum mycelium is forced to initiate apoptosis, thereby inducing and enhancing the total yield of ganoderic acid contained therein. Among them, the control group is a Ganoderma lucidum mycelium which does not add 1-chloro-2,4-dinitrobenzene. As shown in the figure, the addition of 1-chloro-2,4-dinitrobenzene does promote the expression amount (yield) of ganoderic acid.

It can be seen from the above embodiments of the present invention that the application of the present invention has the following advantages:

The invention utilizes the addition of apoptosis inducer to force the cells to undergo apoptosis (Fig. 7), and indirectly induces the increase of the yield of the secondary metabolite of the fungus by the biochemical pathway of apoptosis, thereby increasing the yield. . Accordingly, in the future, it is expected that the secondary metabolites such as triterpenoids or their ganoderic acid, antibiotics, anthuric acid and the like can be mass-produced by the method provided by the present invention, thereby enabling secondary metabolites having many medical effects. High prices and more affordable, close to the people, to provide better Chinese herbal medicine resources.

Although the present invention has been disclosed in the above embodiments, it is not intended to limit the present invention, and the present invention can be modified and modified without departing from the spirit and scope of the present invention. The scope is subject to the definition of the scope of the patent application attached.

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Claims (20)

A method for inducing biosynthesis of secondary metabolites of fungi, which utilizes forcing fungi to induce apoptosis and induces biosynthesis of secondary metabolites, and the method for inducing biosynthesis of secondary metabolites of fungi comprises: providing a fungus Mycelium, and the mycelium is cultured for a predetermined culture time; an apoptosis inducer having an effective concentration is delivered to the mycelium, and cultured for a predetermined induction time, wherein the cell is withered The inducer of death is selected from the group consisting of aspirin and 1-Chloro-2,4-dinitrobenzene, which is effective as a part of aspirin. The concentration is above 0.5 millimolar (mM), the effective concentration of 1-chloro-2,4-dinitrobenzene is above 0.4 millimolar (mM); and the second extraction and purification of the mycelium metabolite. A method of inducing biosynthesis of a secondary metabolite of a fungus according to claim 1, wherein the fungus is Ganoderma lucidum. The method for inducing biosynthesis of a secondary metabolite of fungi according to claim 2, wherein the ganoderma lucidum is deposited in the Bioresource Conservation and Research Center of the Food Industry Development Research Institute under the registration number BCRC 36111. The method of claim 1, wherein the secondary metabolite comprises a Ganoderic acid. The method of claim 1, wherein the apoptosis inducer is aspirin, and the effective concentration is 0.5. Millenol concentration (mM) to 8 millimolar (mM). A method of inducing biosynthesis of a secondary metabolite of fungi according to claim 5, wherein the effective concentration is from 1 millimolar (mM) to 6 millimolar (mM). A method of inducing biosynthesis of a secondary metabolite of fungi according to claim 5, wherein the effective concentration is 4 millimolar (mM). A method of inducing biosynthesis of a secondary metabolite of fungi according to claim 5, wherein the predetermined culture time is from 4 days to 18 days. A method of inducing biosynthesis of a secondary metabolite of a fungus according to claim 5, wherein the predetermined culture time is from 4 days to 16 days. A method of inducing biosynthesis of a secondary metabolite of a fungus according to claim 5, wherein the predetermined culture time is 12 days. A method of inducing biosynthesis of a secondary metabolite of a fungus according to claim 5, wherein the predetermined induction time is from 6 hours to 48 hours. A method of inducing biosynthesis of a secondary metabolite of a fungus according to claim 5, wherein the predetermined induction time is from 12 hours to 36 hours. A method of inducing biosynthesis of a secondary metabolite of a fungus according to claim 5, wherein the predetermined induction time is 24 hours. The method of claim 1, wherein the apoptosis inducer is 1-chloro-2,4-dinitrobenzene, and the effective concentration is 0.4 millimolar (mM). The method of claim 14, wherein the predetermined culture time is from 1 day to 12 days. The method of claim 14, wherein the predetermined culture time is from 2 days to 8 days. A method of inducing biosynthesis of a secondary metabolite of a fungus according to claim 14, wherein the predetermined culture time is 4 days. The method of claim 14, wherein the predetermined induction time is from 1 day to 12 days. The method of claim 14, wherein the predetermined induction time is from 2 days to 8 days. The method of claim 14, wherein the predetermined induction time is 4 days.