TWI385248B - A formula of culturing medium for cordyceps spp. - Google Patents

Description

Formula of Cordyceps Medium

The invention relates to a medium formula of Cordyceps fungi, in particular to a medium formula which can promote the growth of Cordyceps fungi and the formation of metabolites.

Cordyceps spp . belongs to parasitic fungi and has a wide range of host species, including insects, plants, fungi and some arachnids. According to the literature, there are more than 400 species of Cordyceps fungi. A small number of Cordyceps fungi are used as Chinese herbal medicines, and are popular in Asian countries (such as China, Korea, Japan, etc.). Among them, Cordyceps sinensis is recognized as the most effective Cordyceps fungus; the broad-based Cordyceps sinensis is a Cordyceps sinensis Infecting host insects, using the complex formed by the nutrients in the host insects, such as Cordyceps militaris, thistle , and Beauveria bassiana; and the Cordyceps snensis , which is considered by the general public, is mainly Department, as its winter it is like insects living in the soil by the larvae of a child seat Chinese Cordyceps fungus (Cordyceps sinensis) in a bat parasitic insects of the family (Hepialus armoricanus) of (especially grass bat (Oberthur) larvae), to Xia was unearthed and turned into a grass, so it was called Cordyceps sinensis.

The medicinal value of Cordyceps sinensis has been valued by many parties, mainly for the functions of circulatory system, immune activity, respiratory tract, cardiovascular and endocrine system, and it has been confirmed by research that Cordyceps sinensis has many physiological significances such as enhancing immunity and anti-cancer cells. It is also regarded as a therapeutic mushroom in western countries; the main clinical role of Cordyceps sinensis is still unclear, but most scholars believe that Cordyceps contains various components such as cordycepin and adenosine. Etc., is an indicator substance that determines the physiological and biochemical activity of Cordyceps sinensis, and the polysaccharide contained in the fermentation broth has preliminary results in immunomodulation and antitumor activity tests.

However, natural Cordyceps sinensis has strict requirements for growth and requires special geographical environment and climate, so the yield is limited. In recent years, excessive picking and environmental changes have led to the shortage of wild Cordyceps sinensis. Therefore, artificial culture techniques are often used. Mass production in a sterile environment, the conventional method of artificially cultivating Cordyceps sinensis can be divided into two methods: in vivo culture and in vitro culture; the in vivo culture system directly cultures Cordyceps sinensis by artificial inoculation. In parasitic insects, however, the techniques of mass cultivation and artificial inoculation of parasitic insects are still difficult to implement, and often cannot be successfully cultured. In vitro culture methods are inoculated with Cordyceps sinensis into a medium to replace parasitic with artificial formula. Insects, providing the nutrients required for growth of Cordyceps sinensis, according to the different cultures of Cordyceps sinensis, such as YM medium, wheat extract medium or potato carrot medium; the difference in medium formula is mainly from carbon The source or source of nitrogen, Commonly used carbon sources are glucose, maltose, lactose, dextrin, monosaccharide, disaccharide and starch, while the nitrogen source can be yeast powder, soy flour, peptone and wheat bran. In addition, some medium formulas can also be added. Vitamins (such as vitamins B1 and B6), inorganic substances (such as calcium sulfate) or organic salts (such as phosphate) to provide the nutrients needed for the growth of different Cordyceps sinensis; however, most of the growth of Cordyceps sinensis in the formulation medium is not good, so nowadays Artificial culture of Cordyceps sinensis often uses the following three culture forms depending on the culture requirements:

A. Liquid culture form: formulating a liquid medium formula of Cordyceps sinensis with water as a blending solvent, and then inoculation with a Cordyceps sinensis for artificial culture; the cost of using the liquid culture form is lower, the fermented product is easier to collect, and can be A large number of mycelia were obtained in a short time. The mycelium was confirmed by experiments to have similar pharmacological effects with wild Cordyceps sinensis. Therefore, the liquid culture form is gradually adopted by most people, but the disadvantage is that the production scale is small, and some Cordyceps sinensis Poor growth of liquid media also affects the yield of a few filamentous fungi.

For example, the Patent No. 200412371, "Method and Medium for Liquid Culture of Mycelium of Cordyceps Sinensis", discloses a medium and method for liquid culture of mycelium of Cordyceps sinensis, wherein The culture medium is composed of a hydrolysate of a high molecular sugar agricultural product as a nutrient substrate to culture the mycelium of Cordyceps sinensis; however, although the liquid culture method uses a high molecular sugar agricultural product as a carbon source of the culture medium, the cultivated winter insect grass is cultivated. The fermentation yield of the bacteria has not been significantly improved, and in order to enable the high molecular sugar agricultural product to be directly used by the mycelium of the cultivated Cordyceps sinensis, the high molecular sugar agricultural product must be transformed by a multi-step hydrolysis treatment. In order to reduce the sugar, the cultivation process is more complicated.

B. Solid-state culture: a gel is added to a liquid medium formulation, and the medium is formulated into a solid gel-like culture medium at room temperature, and is also inoculated with Cordyceps sinensis for fermentation production. The commonly used culture medium has Maltaceae (MEA), potato glucosyl (PDA) or yeast acacia (YEA), etc.; in general, the solid culture form can provide the growth of Cordyceps sinensis due to its culture medium, which is beneficial to the growth. The growth of fruiting bodies, however, the processing and operation of solid state culture is complicated and time consuming, and the relevant culture conditions (such as temperature, humidity and moisture) are not easy to control and the medium has a high chance of contaminating the bacteria, which is easy to cause production and production. difficult.

For example, the Patent Case of the Republic of China Announcement No. I231825 "a method for solid-state fermentation of reproductive fungi" discloses a solid fermentation method suitable for cultivating fungi such as Cordyceps sinensis, Brazilian mushroom and Ganoderma lucidum. The culture method first inoculates a fungus into a solid medium to grow, and then sequentially transfers to a liquid medium and a solid fermentation medium to facilitate control of the temperature, moisture, gas supply and fermentation of the culture environment. The product; however, the operation procedure of the solid culture form is too complicated, the culture period is long, and the yield of the metabolite of the cultured fungus is not significantly increased, and the quality of the fermentation product is not improved.

C. Semi-solid culture: refers to a liquid medium formula containing a solid nutrient substrate. The solid medium is mostly rice, wheat and other cereals. It can be used as a nutrient source for cultured bacteria, and can be used for cell retention and maintenance. Liquid fermentation fermentation under the characteristics of climbing growth to improve the production process of fungi; such as the Republic of China Announcement No. I242044 "Method for cultivating filamentous fungi using solid nutrient substrate suspension" patent, reveals a solid nutrient matrix The fermentation broth is inoculated into the fermentation broth for production, and has the advantages of solid culture and liquid fermentation; however, the semi-solid medium still cannot completely improve both the solid culture and the liquid culture. Disadvantages, the artificial culture of Cordyceps sinensis and the production of its fermentation products still cannot achieve an optimal production state.

From the above, the artificial in vitro culture of Cordyceps sinensis is mainly to inoculate Cordyceps sinensis into the culture medium for cultivation, although the application of the formulation medium has developed various formula components (different carbon sources, nitrogen) for the growth difference of various Cordyceps sinensis. Source), but generally has the disadvantages of poor culture effect, such as cell growth and fermentation products, so it is necessary to adopt different culture types (liquid, solid and semi-solid culture) according to the purpose of industrial culture, or to develop some improved The culture method is used to enhance the growth and fermentation of Cordyceps sinensis, but it often makes the operation process complicated and can not make the effect remarkable. Therefore, it is necessary to improve the medium formula of the fungus fungus to improve the progress of the Cordyceps true artificial culture industry. .

The invention mainly relates to the improvement of the conventional artificial culture technique of Cordyceps fungi, and provides a formula of Cordyceps culture medium, the main purpose of which is to promote the growth state of Cordyceps fungi in artificial culture environment.

A second object of the present invention is to provide a formulation of Cordyceps culture medium to enhance the yield of the fermentation product of Cordyceps fungi in an artificial culture environment.

A further object of the present invention is to provide a formulation of a Cordyceps culture medium for enhancing the yield of polysaccharides of Cordyceps fungi in an artificial culture environment.

A further object of the present invention is to provide a formulation of a Cordyceps culture medium for enhancing the mycelial yield of Cordyceps fungi in an artificial culture environment.

In order to achieve the foregoing object, the technical means used in the present invention comprises: a formulation of a Cordyceps medium comprising: a carbon source having a concentration of 1 to 6% by weight; a nitrogen source of 0.85 to 1.5%; ~12% alginate; and a 86.2~96.2% blending solvent.

The above and other objects, features, and advantages of the present invention will become more apparent from the description of the preferred embodiments of the invention. The alginate is added to the artificial culture medium of the Cordyceps fungus, and is formulated into a liquid fermented Cordyceps culture medium, which can effectively increase the mycelial growth, polysaccharide yield and yield of the fermented product of the Cordyceps fungus.

In the present embodiment, a strain of Cordyceps militaris is inoculated to the Cordyceps culture medium of the present invention, and the growth state and the fermentation state of the wild Cordyceps militaris mycelium are good; the Cordyceps culture medium is composed of a concentration by weight 1~6% carbon source; a 0.85~1.5% nitrogen source; a 2~12% alginate; and a 86.2~96.2% blending solvent are uniformly mixed and prepared.

The carbon source of the Cordyceps culture medium of the present invention comprises a carbon source (such as glucose, fructose, lactose or maltose, etc.), which is selected from a high utilization sugar molecule, and is used as the main nutrient substrate for the artificial culture of the Cordyceps militaris. Providing the main nutrients required for the growth of the Cordyceps militaris; and the nitrogen source contained in the Cordyceps culture medium comprises a fixed nitrogen source (such as yeast extract or maltose extract) and a nitrogen source (such as peptone). Or corn flour, etc.), also provides the basic nutrients needed for the growth of Cordyceps militaris.

The Cordyceps culture medium of the present invention is prepared by uniformly mixing and dissolving the carbon source, the nitrogen source and the alginate by using water as a preparation solvent, wherein the alginate may be added in the form of solid, liquid or powder, and the addition of alginate The texture of the Cordyceps culture medium can be made thicker but still kept in a liquid state, so that the inoculated Cordyceps militaris has a stronger binding force, and the liquid culture fermentation form of the Cordyceps culture medium is beneficial to the Cordyceps militaris. The growth of mycelium has a promoting effect on the nutrient acquisition and metabolic reaction of Cordyceps militaris. In addition, the Cordyceps culture medium maintains the good growth of the Cordyceps militaris to be inoculated, and an additional weight percentage of 0.05 can be added. ~0.1% of inorganic substances (such as potassium phosphate or magnesium sulfate), and preferably adjust the pH to pH 6; the detailed formulation of the Cordyceps medium of the present invention is shown in the following table:

In order to confirm that the Cordyceps culture medium of the present invention can effectively promote the mycelial growth of Cordyceps fungi, the Cordyceps culture medium of the present invention is formulated according to the above formula, and the strain of Cordyceps militaris is introduced into the Cordyceps genus of the present invention in an inoculation loop. The medium is a test group (1); and a common medium is prepared by using a carbon source (glucose) and a nitrogen source (maltose extract, peptone and yeast extract) having the same composition and ratio as the Cordyceps medium of the present invention. However, without adding the alginate of the present invention, the strain of the Cordyceps militaris was inoculated in the same manner as the control group (2); the inoculation liquid of the Cordyceps militaris in the above test group (1) and the control group (2) was placed in a constant temperature. The culture incubator is cultured in a culture environment of 20 to 25 ° C at a stirring speed of 150 rpm for 14 to 30 days, and the Cordyceps culture medium of the present invention and the growth state of the Cordyceps militaris cultured by the common medium are periodically observed and recorded during the culture period. State, the results are shown in Figure 1.

As shown in Fig. 1, the Cordyceps militaris cultured in the test group (1) was observed to have a positive growth state (+20%) on the third day after inoculation, and the test group (1) Cordyceps militaris remained in a good growth state (>10%) during the culture period (30 days); while the control group (2) Cordyceps sinensis showed a negative growth state on the 6th day after inoculation, and The situation of negative growth is more pronounced as the incubation time increases (at day 30). The results show that the Cordyceps culture medium of the present invention can promote the growth of Cordyceps militaris because of the alginate component, and the growth state of the medium is negative from positive to positive. To the growth, it was confirmed that the alginate of the present invention has a clear and remarkable promoting effect on the artificial culture of the Cordyceps militaris.

The Cordyceps militaris used in this embodiment, also known as Cordyceps militaris or Bombyx mori, has physicochemical functions such as sedative, antiasthmatic, antiarrhythmia and anti-fatigue, and enhances immunity; Cordyceps militaris has a special chemical composition. Such as mannitol, adenosine, cordycepin and cordyceps polysaccharides, it has the effect of health care, wherein the cordyceps polysaccharide is one of the main active substances, and has anti-tumor, anti-aging, anti-oxidation, anti-virus, and descending Blood sugar and blood fat reduction and other functions. At present, although artificial cultivation of Cordyceps militaris has been applied to a considerable extent, due to the difficulty of growth, there is still a technical bottleneck for controlling the production of mycelium of Cordyceps militaris and the production of useful metabolites; The mycelium growth of the Cordyceps militaris has a beneficial effect, and has a better effect on the yield and activity of the polysaccharide of the Cordyceps militaris, in order to confirm that the Cordyceps culture medium of the present invention can promote the yield and activity of the polysaccharide of the Cordyceps militaris. The above-mentioned Cordyceps militaris culture test is again operated, and an appropriate amount of the culture liquid is taken periodically (every 3 days) during the culture, and the impurities are removed by a centrifugal filtration method, and the content of the polysaccharide in the culture liquid is analyzed by a phenol sulfuric acid method. The yield of polysaccharides produced by Cordyceps militaris cultured in the test group (1) and the control group (2) was compared, and the results are shown in Fig. 2.

As shown in Fig. 2, the Cordyceps militaris cultured in the test group (1) is capable of detecting polysaccharide formation in the culture broth after inoculation, and the yield thereof is as high as 0.45 (g/unit liter). In the culture process (0-6 days), the yield of the polysaccharide produced by the test group (1) was maintained at 0.4 (grams per unit volume liter), and the produced polysaccharide had good activity; The Cordyceps militaris cultivated in the group (2) was found to have a higher polysaccharide yield from the culture broth on the third day after inoculation, about 0.3 (g/unit liter liter), but the control group (2) The content of polysaccharides produced by the cultured Cordyceps militaris was still significantly lower than that of the test group (1), and the activity was also low. The results show that the Cordyceps culture medium of the present invention can promote the production of polysaccharides with more activity and better activity due to the inclusion of alginate components, and the polysaccharides of the general medium without alginate added. The content was significantly improved, and it was confirmed that the alginate of the present invention has an effect of promoting the production of its metabolites for the artificially cultivated Cordyceps militaris.

As described above, the Cordyceps culture gene of the present invention contains alginate component, which can effectively increase the mycelium production amount, metabolite yield and metabolite activity of the cultured Cordyceps fungus, and expand the culture of the Cordyceps fungus. And the use of the fermented product is helpful; in addition, the high-yield and high-activity metabolite can be obtained by the Cordyceps culture medium of the present invention, thereby separating the active ingredient with higher purity for the medical products related to Cordyceps fungi. The development and research of the active ingredients in various aspects such as anti-oxidation, anti-tumor and anti-aging are of great significance, which is conducive to the further development of the research.

The invention relates to a Cordyceps culture medium which is prepared by adding a brown algae component to a general medium formula (containing a carbon source, a fixed nitrogen source and a nitrogen source, etc.), so that the texture of the cordyceps medium is thicker, and the method is provided. Cordyceps fungi are required for growth, and have the effect of promoting the growth of Cordyceps fungi.

The invention relates to a Cordyceps culture medium, which is characterized in that a brown algae component is added to a general medium formula (containing a carbon source, a fixed nitrogen source and a nitrogen source, etc.) to maintain a liquid fermentation state, which can be improved. The yield and activity of the metabolites of Cordyceps fungi have the effect of promoting the artificial culture technique of Cordyceps fungi.

The formula of the Cordyceps culture medium of the present invention can directly control a plurality of culture advantages such as the mycelium yield and the amount of polysaccharide production of the Cordyceps fungus, and has the advantages of simple operation and low cost. .

While the invention has been described in connection with the preferred embodiments described above, it is not intended to limit the scope of the invention. The technical scope of the invention is protected, and therefore the scope of the invention is defined by the scope of the appended claims.

1‧‧‧Test group

2‧‧‧Control

Figure 1: Schematic diagram of the growth state of Cordyceps culture medium of the present invention

Figure 2: Schematic diagram of the metabolite production of the Cordyceps medium of the present invention

Claims (4)

The formula of Cordyceps culture medium comprises: glucose concentration of 1 to 6% by weight; nitrogen source of 0.85 to 1.5%; alginate of 2 to 12%; and formulation solvent of 86.2 to 96.2%. A formulation of Cordyceps culture medium according to claim 1, wherein the nitrogen source is selected from the group consisting of a malt extract, a yeast extract, and a peptone. Cluster. A formulation of a Cordyceps culture medium according to the first aspect of the patent application, wherein the Cordyceps medium has a pH of 6. According to the formula of the Cordyceps culture medium according to the first aspect of the patent application, wherein the solution medium of the Cordyceps culture medium is water.