TWI774104B - Culture medium and culturing method of polypore mycelial pellets - Google Patents

Abstract

The invention provides a culture medium and culturing method of polypore mycelial pellets, in which the characteristic is that a culture medium including potato dextrose medium solution and unhydrolyzed animal protein is used to perform a liquid state fermenting step on a polypore mycelium, thereby increasing yields of the mycelial pellets.

Description

Culture liquid and method for culturing polyporous fungus mycelial balls

The present invention relates to a method for culturing fungi, in particular to a culture solution and method for culturing polyporous fungus mycelial balls.

Many species of polypores have a variety of effective substances (such as: triterpenoids, phenolic compounds, polyketides, polysaccharides and sterols, etc.) and have medicinal value (such as: Ganoderma lucidum, Yunzhi, Antrodia camphorata, Poria, etc.). The above-mentioned effective substances can exist not only in the fruit body, but also in the mycelium, so the mycelium also has medicinal value.

According to the type of medium, the methods of culturing medicinal fungi can be roughly divided into two categories: solid-state fermentation and liquid fermentation. Among them, solid-state fermentation is mostly used to produce fruiting bodies, and liquid culture is mostly used to produce mycelium. Compared with solid fermentation, the conditions of liquid fermentation are easier to control, and the production cycle of mycelium is shorter than that of fruiting bodies, so liquid culture has the potential for large-scale production.

According to the composition of the culture medium and the culture method, the mycelium can form a filamentous or spherical shape in the culture medium, and the shape of the mycelium can affect the total yield. Specifically, filamentous mycelium increases the viscosity and non-Newtonian Rheology, causing poor mass transfer (such as oxygen or temperature transfer), resulting in lower yields. On the contrary, under the condition of low density, spherical mycelium (pellets) does not have the above problems, so by adjusting the culture method, the shape of the mycelium can be changed, thereby increasing the total yield of mycelium.

Therefore, there is an urgent need for a method for culturing mycelial balls to improve the total yield of mycelium.

Therefore, one aspect of the present invention is to provide a method for culturing mycelial balls, which is to add non-hydrolyzed animal protein to the culture solution to increase the number and biomass of the mycelial balls.

According to the above aspect of the present invention, a method for culturing mycelial balls is provided. The above-mentioned method can, for example, use a culture solution to perform a liquid fermentation step on the mycelium of Polyporus to obtain mycelial balls, wherein the culture solution can include but is not limited to potato dextrose broth (PDB) and non-porous dextrose broth. Hydrolyzed animal protein (unhydrolyzed animal protein), and the content of the unhydrolyzed animal protein in the PDB may be, for example, less than or equal to 1 weight percent (wt%).

According to the above-mentioned embodiment of the present invention, the non-hydrolyzed animal protein can be derived from egg yolk, for example.

According to the above-mentioned embodiment of the present invention, the culture medium can optionally contain 0.25wt% to 0.75wt% of yeast extract.

According to the above-mentioned embodiment of the present invention, before performing the liquid fermentation step, the above-mentioned method can optionally include heat-inactivating the culture medium. (heat-inactivated) processing.

According to the above-mentioned embodiments of the present invention, the polypore bacteria may include, but are not limited to, Fomitopsis spp. and Ganoderma spp.

According to the above-mentioned embodiment of the present invention, the temperature of the liquid fermentation step may be, for example, 23° C. to 29° C., and the rotation speed of the liquid fermentation step may be, for example, 100 rpm to 150 rpm.

Another aspect of the present invention is to provide a culture solution for culturing mycelial balls, which can be applied to the above-mentioned method for culturing mycelial balls.

According to the above aspect of the present invention, a culture solution for culturing mycelium balls is provided, which is characterized in that the potato dextrose culture solution (PDB) contains less than or equal to 1wt% of non-hydrolyzed animal protein.

According to the above-mentioned embodiment of the present invention, the culture medium can optionally contain 0.25wt% to 0.75wt% of yeast extract.

According to the above-mentioned embodiment of the present invention, the non-hydrolyzed animal protein can be derived from egg yolk, for example.

According to the above-described embodiments of the present invention, the culture medium may be, for example, heat inactivated.

By applying the method for culturing mycelial balls of the present invention, the number and biomass of the mycelial balls can be significantly increased, thereby increasing the total yield of the mycelium.

601,603: Straight

In order to make the above and other objects, features, advantages and embodiments of the present invention more clearly understood, the detailed description of the accompanying drawings is as follows:

[ Fig. 1 ] is a bar graph showing the relative contribution of different components to the change in the number of mycelial balls according to an embodiment of the present invention.

[FIG. 2A] to [FIG. 2E] show the results of the liquid fermentation step with the culture solution containing 0 wt %, 0.25 wt %, 0.5 wt %, 0.75 wt % and 1 wt % egg yolk powder according to an embodiment of the present invention photo.

[ Fig. 3 ] is a bar graph showing the content of egg yolk powder versus the number of mycelial balls according to an embodiment of the present invention.

[ Fig. 4 ] is a bar graph showing the content of egg yolk powder versus the diameter of mycelial balls according to an embodiment of the present invention.

[ Fig. 5 ] is a bar graph showing the content of egg yolk powder versus biomass according to an embodiment of the present invention.

[ Fig. 6 ] is a bar graph of the number of mycelial balls cultured by different strains in a culture solution with or without egg yolk powder according to an embodiment of the present invention.

A

11%)；數值範圍若未界定下限值(如低於0.2%的B，或0.2%以下的B)，則皆指其下限值可能為0(即0%

B

0.2%)。上述用語是用以說明及理解本發明，而非用以限制本發明。 References herein to the singular forms "a,""an," and "the" include plural references unless the context clearly dictates otherwise. The numerical range (such as A from 10% to 11%) includes the upper and lower limit values unless otherwise specified (ie 10%

A

11%); if the value range does not define the lower limit (such as B below 0.2%, or B below 0.2%), it means that the lower limit may be 0 (ie 0%

B

0.2%). The above terms are used to describe and understand the present invention, but not to limit the present invention.

The method for culturing mycelial balls (pellets) provided by the present invention utilizes unhydrolyzed animal protein (unhydrolyzed animal protein) The potato dextrose broth (PDB) of the protein) was subjected to a liquid fermentation step on the mycelium of the polypore fungus to increase the number and biomass of mycelial balls.

The "mycelium" is a collection of hyphae obtained by culturing multicellular fungi through a liquid fermentation step. The shape of the mycelium is variable, it can be a filamentous mycelium with freely dispersed mycelium, or a spherical mycelium with a highly entangled mycelium (ie, mycelium ball). Cell wall composition, strain size, growth rate, carbon/nitrogen ratio of the medium, pH, temperature, ion concentration, and shear force.

The "polypore" refers to a group of basidiomycetes with basidiomycetes growing in the pore-shaped fruit layer. Generally speaking, the fruiting body of Polyporus is harder and less prone to spoilage, hence the common name of Hard Mushroom. According to the modern fungal classification system, polyporous fungi include Polyporales and Hymenochaetales, among which Polyporales include Ganoderma, Fomitopsidaceae and Polyporaceae (Fomitopsidaceae). Polyporaceae) and so on. In one embodiment, the polyporous bacteria may include Fomitopsis and Ganoderma. In one embodiment, the polypore comprises Ganoderma tropicum, Ganoderma gibbosum, Fomitopsis pinicola, or other strains of the same genus Ganoderma or Pseudomonas.

The PDB can be formulated, for example, with a conventional formula (fresh potato and glucose), or by using commercially available PDB powder plus water.

The "non-hydrolyzed animal protein" uses animal protein but excludes Except for the use of enzymatic hydrolysis. In one embodiment, the non-hydrolyzed animal protein can be derived, for example, from the egg yolk of any bird or reptile, such as egg, duck, quail or lizard eggs. In one embodiment, the above-mentioned dosage form of egg yolk can be, for example, powder after spray-drying the egg, such as egg yolk powder.

Experiments have confirmed that non-hydrolyzed animal protein can effectively increase the number and biomass of mycelial balls, thereby increasing the total yield of mycelium. The number and biomass of mycelial balls increase with the increase of the content of non-hydrolyzed animal protein. However, if the content of non-hydrolyzed animal protein is greater than 1 weight percent (wt%), not only the number and biomass of mycelial balls cannot be increased, but also pollution may be caused. In one embodiment, the content of non-hydrolyzed animal protein in PDB is 0.25 wt %.

In one embodiment, the above-mentioned culture medium may optionally contain yeast extract. The amount of yeast extract used is not limited, and can be, for example, 0.25 wt % to 0.75 wt %.

In one embodiment, before the liquid fermentation step, it may optionally include heat-inactivating the culture medium. The heat inactivation treatment can be carried out by any known heat inactivation method. In one embodiment, the heat inactivation treatment may be performed, for example, at a temperature of 100° C. to 150° C. and a pressure of 1.0 kg/cm ² to 1.5 kg/cm ² for 10 to 30 minutes.

In one embodiment, the temperature of the liquid fermenting step can be performed at a temperature at which polypore bacteria prefer to grow, such as 23°C to 29°C. In one embodiment, the rotation speed of the liquid fermentation step is 100 rpm to 150 rpm, so that the mycelium can form a mycelium ball.

Several embodiments are used below to illustrate the application of the present invention, but they are not intended to limit the present invention. Those with ordinary knowledge in the technical field of the present invention can make various changes and modifications without departing from the spirit and scope of the present invention. retouch.

Embodiment 1. Analyze the influence of different components on the number of mycelial balls

Taguchi analysis method was used to analyze the effect of potato flour [manufacturer: Basic American Foods; product name: delicious quick-cooking potato flour], glucose, yeast extract and egg yolk powder on the number of mycelial balls respectively. Relative percentage contribution assessment. First, using the formula in Table 1, water was added and a heat inactivation step (temperature: 121° C., pressure: 1.2 kg/cm ² , time: 25 minutes) was respectively added to prepare the culture solutions of groups 1 to 9. Next, liquid fermentation was performed on the mycelium of Ganoderma tropicum (the diameter of 0.8cm, the bacterial mass was obtained after culturing at 25°C using PDA culture agar) at a rotational speed of 130 rpm at 25°C. The procedure was carried out for 7 days, and the number of mycelial balls was counted. The above tropical Ganoderma lucidum can be collected from Dawu Mountain in Pingtung, Taiwan [GeneBank No. MK007291.1] or other strains of tropical Ganoderma lucidum, such as deposited in the Bioresourse Collection and Research Center (BCRC) The strains with accession numbers BCRC 35346, BCRC 36209, BCRC 37032, BCRC 37041, BCRC 37065, BCRC 37067 or BCRC 37122. The results are listed in Table 1. The above-mentioned egg yolk powder is formed by spray drying the fresh egg yolk.

Then, the contribution of different components is calculated, and the results are shown in Figure 1. 1 is a bar graph showing the relative contribution of different components to the change in the number of mycelial balls according to an embodiment of the present invention, wherein the horizontal axis represents the components, and the vertical axis represents the sum of the contributions of all components as 100%. The relative contribution of each component. As shown in Figure 1, the relative contribution of egg yolk powder is the highest, indicating that egg yolk powder is the component that has a better effect on the number of mycelial balls among the above four components.

Embodiment 2. Evaluate the influence of egg yolk powder content on the number of Ganoderma lucidum mycelial balls

Use commercially available potato dextrose broth (potato dextrose broth, PDB) powder to add water to prepare 1-fold PDB. Next, egg yolk powder is added to the PDB to prepare a culture solution containing 0.25wt%, 0.5wt%, 0.75wt% and 1wt% egg yolk powder respectively, and a heat inactivation step is performed, wherein the egg yolk powder will be deactivated after the heat inactivation step. Suspended in culture medium. The above-mentioned liquid fermentation step was performed on the mycelium of Ganoderma lucidum tropicalis in a 250-mL conical flask using 100 mL of culture medium to produce mycelial balls. put the liquid After the fermentation step, the culture solution was poured into a petri dish, as shown in Figure 2A to Figure 2E. FIG. 2A to FIG. 2E show that according to an embodiment of the present invention, it contains 0 wt % ( FIG. 2A ), 0.25 wt % ( FIG. 2B ), 0.5 wt % ( FIG. 2C ), 0.75 wt % ( FIG. 2D ) and 1 wt % (FIG. 2E) A photograph of the result of the liquid fermentation step performed on the broth of egg yolk powder. As shown in Fig. 2A to Fig. 2E, the number of mycelial balls increased with the increase of egg yolk powder content.

Next, the number of mycelial balls per unit volume was calculated and the results are shown in FIG. 3 . 3 is a bar graph showing the content of egg yolk powder versus the number of mycelial balls according to an embodiment of the present invention, wherein the horizontal axis is the content of egg yolk powder, the vertical axis is the number of mycelial balls per unit volume, and different letters (a, b and c) indicate that there are significant differences between groups (P<0.05) after statistical analysis using the least-significant difference (LSD) method. As shown in FIG. 3 , the number of mycelial balls per unit volume obtained from the broth containing egg yolk powder was significantly higher than that of PDB without the addition of egg yolk powder. In addition, the number of mycelial balls per unit volume increased with the increase of egg yolk powder content, indicating that egg yolk powder had a dose dependent effect on the number of mycelial balls.

Then, the spherical diameter of the mycelial balls was analyzed using the commercially available software IMAGE J, and the results are shown in FIG. 4 . 4 is a bar graph showing the content of egg yolk powder versus the diameter of mycelial balls according to an embodiment of the present invention, wherein the horizontal axis represents the content of egg yolk powder, the vertical axis represents the diameter of the ball, and the same letter (a) represents the use of the least significant difference There was no statistically significant difference (P<0.05) after statistical analysis. As shown in Figure 4, the mycelial balls cultured with different contents of egg yolk powder have no statistically significant difference in ball diameter, wherein the ball diameter is 0.33 cm to 0.36 cm. In detail, the number of mycelial balls increased with the increase of egg yolk powder content, and when the number of mycelial balls was small, the difference in the diameter of the mycelial balls was large (large number of variation), but when the number of mycelial balls was large , the difference in the diameter of the mycelial ball becomes smaller (the number of variation is small).

Mycelium balls were dried and their dry weight was measured to evaluate the effect of egg yolk powder content on biomass, and the results are shown in Figure 5. 5 is a bar graph showing the content of egg yolk powder versus biomass according to an embodiment of the present invention, wherein the horizontal axis is the egg yolk powder content, the vertical axis is the biomass per unit volume, and different English letters indicate the use of the least significant difference method After statistical analysis, there were significant differences among the groups (P<0.05). As shown in Figure 5, compared with PDB without egg yolk powder, the biomass of mycelial balls cultured with 0.25wt% egg yolk powder was significantly higher, indicating that egg yolk powder can effectively increase the biomass. Secondly, the biomass increased with the increase of egg yolk powder content, showing a dose-dependent effect of biomass and egg yolk powder.

The following uses a culture medium with a specific content of egg yolk powder to evaluate its effect on increasing the number of other polyporous fungus mycelial balls.

Example 3. Evaluation of the influence of the culture solution of Example 2 on the number of polyporous fungus mycelial balls

A culture solution containing or not containing 0.25 wt% egg yolk powder was prepared respectively, and the above-mentioned liquid fermentation step was performed on the mycelia of the polypore fungi listed in Table 2. Table 2 shows the scientific name of the bacterial species, the corresponding GeneBank number of the 18S rRNA sequence and the number of days of cultivation, wherein the bacterial species 1 to 3 were collected from Dawu Mountain in Pingtung, Taiwan, and were respectively Ganoderma gibbosum ), Ganoderma sp. and Pseudomonas Fomitopsis pinicola, but strains 1 to 3 can also be other strains of the same strain, for example, strain 1 can be Ganoderma stipitate with deposit number BCRC 36441, strain 2 can be It is the strain with the deposit number of BCRC 37046 or BCRC 37047, and the strain 3 can be BCRC 32527, BCRC 35303 or the Pseudomonas pineapple described in the Korean patent KR 100778941 B1 published on November 28, 2007, and its depository institution It is the Korean Agricultural Genetic Resources Collection (Korean Agricultural Culture Collection, KACC), and the deposit number is KACC 93042 P.

The liquid fermentation step was carried out, and the number of mycelial balls was counted, and the results were listed in Figure 6. 6 is a bar graph of the number of mycelial balls cultured with different bacterial species in a culture solution with or without egg yolk powder according to an embodiment of the present invention, wherein the horizontal axis represents bacterial species, and the vertical axis represents mycelium per unit volume number of balls, and bars 601 and 603 indicate no and added egg yolk powder, respectively. As shown in FIG. 6 , compared with the PDB without egg yolk powder, the number of mycelial balls cultured in the culture solution with egg yolk powder was higher, indicating that egg yolk powder can increase the number of mycelial balls of foramen.

It can be seen from the above examples that the method for culturing mycelial balls of the present invention can increase the yield of mycelium by increasing the number and biomass of mycelial balls of foraminifera.

It should be added that although the present invention takes a specific bacterial species, a specific strain, a specific concentration and/or a specific analysis method as an example, it is explained that the method for culturing the mycelial ball of the present invention can improve the mycelial ball and the biomass. However, any person with ordinary knowledge in the technical field to which the present invention belongs will know that the present invention is not limited to this. Other species, other strains, other concentrations, and/or other analytical methods illustrate efficacy.

Although the present invention has been disclosed above with several embodiments, it is not intended to limit the present invention. Anyone with ordinary knowledge in the technical field to which the present invention belongs, without departing from the spirit and scope of the present invention, can make various Therefore, the scope of protection of the present invention should be determined by the scope of the appended patent application.

Claims (8)

A method for culturing polyporous fungus mycelium balls, which is to use a culture solution to perform a liquid fermentation step on one of the mycelium of polypore bacteria to obtain a mycelial balls, characterized in that the culture solution is made of potato dextrose Culture broth (potato dextrose broth, PDB), 0.25wt% to 0.75wt% yeast extract and non-hydrolyzed egg yolk, thereby increasing a number and a biomass of the mycelial ball, and reducing the mycelial ball. A variation of a ball diameter, wherein the culture medium is subjected to a heat-inactivated treatment, and a content of the non-hydrolyzed egg yolk in the PDB is 0.25 wt % (wt %) to 1 wt %. The method for culturing polypore mycelium balls according to claim 1, wherein the non-hydrolyzed egg yolk is derived from birds and/or reptiles. The method for culturing polyporous fungus mycelium balls as claimed in claim 1, wherein the egg yolk is a powder obtained by a spray drying process. The method for culturing polyporous fungus mycelial balls as claimed in claim 1, wherein the polyporous fungus comprises Fomitopsis spp. and Ganoderma spp. The method for culturing polypore mycelium balls as claimed in claim 1, wherein a temperature of the liquid fermentation step is 23°C to 29°C, and a rotation speed of the liquid fermentation step is 100rpm to 150rpm. A culture solution for culturing polyporous fungus mycelial balls is characterized in that the culture solution is composed of potato dextrose culture solution (PDB), 0.25wt% to 0.75wt% of yeast extract and 0.25wt% to 1wt% of non-hydrolyzed It is composed of egg yolk, and the culture medium is treated with heat inactivation. The culture solution for culturing polyporous fungus mycelial balls according to claim 6, wherein the non-hydrolyzed egg yolk is derived from birds and/or reptiles. The culture solution for culturing polyporous fungus mycelium balls according to claim 6, wherein the egg yolk is a powder obtained by a spray drying process.

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