CN104480022A - Collecting method of liquid-cultured fungus mycelia - Google Patents

Abstract

The invention discloses a method for collecting fungal mycelium in liquid culture. Aiming at the shortcomings of existing methods such as poor collection effect, cumbersome and time-consuming operation, and high cost, the collection method disclosed in the present invention adopts a filtration method and uses a steel wire mesh as a filter membrane. The method for collecting fungal mycelium in liquid culture of the present invention not only has a low water content in the obtained mycelium, but also has a wide application range, and is suitable for viscous or clear fermentation liquid. The method provided by the invention is simple and quick to operate, the filter membrane can be reused, the cost is low, the method is energy-saving, low-carbon and environment-friendly, therefore, it is worth popularizing and using.

Description

A kind of collection method of liquid culture fungal mycelium

technical field

The invention relates to a method for collecting liquid-cultured fungal mycelia.

Background technique

In the research of the extraction of total DNA, RNA and protein of fungal mycelium or the research of fungal fermentation formula and parameter exploration, researchers need to carry out liquid culture and proliferation of mycelium, and then separate the mycelium from the fermentation broth. At present, high-speed centrifugation and filtration are often used to separate the mycelium from the fermentation broth. However, both separation methods have certain problems.

For the high-speed centrifugation method, on the one hand, because the mycelium of some fungal strains in liquid culture is relatively viscous and in the state of large particles of suspension, it is difficult to completely compact the mycelium even if a high centrifugal speed is used. Settling to the bottom of the centrifuge tube, the separation effect is poor; on the other hand, the water inside the mycelium ball particles is also difficult to remove by centrifugation, and there are also problems such as high cost of the centrifuge, cumbersome and time-consuming operation.

For the filtration method, currently include the method of vacuum filtration using filter paper and the ordinary gauze filtration method. For the filter paper vacuum filtration method, on the one hand, there are also high and viscous fermented liquids of certain fungal strains, which are difficult to obtain. The problem of drier mycelium, on the other hand, for such a fermentation liquid, the micropores of the filter paper are easily blocked by the fermentation liquid, so it is difficult to filter in the future, and the filter paper must be replaced continuously, and the filter paper is easy to be scratched when it is wet , so the mycelium adsorbed on the filter paper is also difficult to remove completely; for the gauze filtration method, since the gauze is easy to stain the mycelium, it is easy to cause cross-contamination of the mycelium between different samples in the case of a large number of samples. Repeated use, thereby causing unnecessary waste, and when using gauze to filter, the mycelia will easily leak out if the number of gauze layers is small, and the mycelia will easily remain between each layer of gauze and in the holes of the gauze if the number of layers is large, which will easily cause waste and waste of samples. data error.

Contents of the invention

In view of the shortcomings of the above two methods of poor collection effect, cumbersome operation, time-consuming, high cost, etc., the present invention provides a method for collecting liquid to cultivate fungal mycelium with good effect, wide application range, simple, quick, low cost, energy saving and environmental protection method.

The present invention achieves the above object through the following schemes:

The invention discloses a method for collecting liquid cultured fungal mycelia, which adopts a filtration method and uses a steel wire mesh as a filter membrane.

Among them, the steel wire mesh used as a filter membrane can be reused after being washed with sterile water, which is energy-saving and environmentally friendly, and experiments have proved that it will not cause cross-contamination of mycelium between different samples.

Preferably, the above-mentioned steel wire mesh is a 150-250 mesh steel wire mesh. the

As further, above-mentioned wire mesh is 200 mesh wire mesh, and above-mentioned fungus is shiitake mushroom or flat mushroom or black fungus.

Preferably, the above-mentioned filtration method is a vacuum suction filtration method.

In a preferred embodiment, a method for collecting fungal mycelium in liquid culture comprises making a 150-250 mesh steel wire mesh as the filter membrane of the funnel, pouring the fungal fermentation broth into the funnel of the suction filtration system, and starting the suction filtration , when there is no drop in the funnel, the fungal mycelium on the steel wire net is collected, and the collection of the liquid culture fungal mycelium is completed.

In another preferred embodiment, a method for collecting liquid cultured fungal mycelia comprises making a 150-250 mesh steel wire mesh as a filter membrane of a funnel, pouring the fungal fermentation broth into the funnel of the suction filtration system, and starting to pump Filter until there is no liquid drop in the funnel, collect the fungal mycelium on the steel wire net and the fungal fermentation broth in the suction filter bottle of the suction filtration system respectively, and complete the collection of the liquid culture fungal mycelium.

Further preferably, the suction filtration system includes a vacuum pump, a suction filtration flask connected to the vacuum pump, and a funnel installed on the suction filtration flask.

Further preferably, the above-mentioned funnel is a Buchner funnel.

In another specific preferred embodiment, the specific steps of making the 150-250 mesh steel mesh as the filter membrane of the funnel can be: cutting the 150-250 mesh steel mesh into a circle consistent with the caliber of the funnel, and then the steel mesh Put it in the funnel, press and fit into shape.

In the process of collecting fungal mycelium in liquid culture, the inventor unexpectedly found that when using steel wire mesh, especially 150-250 mesh steel wire mesh instead of conventional filter paper for vacuum filtration, the water content reduction rate of the obtained mycelium can be as low as 15%-20% or less, and at the same time, the operation is simple, the separation speed is fast, and the steel wire mesh can also be reused without causing cross-contamination of mycelium between different samples.

The method for collecting fungal mycelium in liquid culture of the present invention not only has a low water content in the obtained mycelium, but also has a wide application range, and is suitable for viscous or clear fermentation liquid. The method provided by the invention is simple and quick to operate, the filter membrane can be reused, the cost is low, the method is energy-saving, low-carbon and environment-friendly, therefore, it is worth popularizing and using.

Detailed ways

The present invention will be further described below in conjunction with specific examples.

Example 1:

The invention discloses a method for collecting fungal mycelium in liquid culture, which adopts a conventional filtration method, and adopts a steel wire mesh, especially a 150-250 mesh steel wire mesh instead of conventional filter paper or gauze as a filter membrane. When using this method to collect the mycelium of fungal liquid culture, the fungal fermentation liquid is poured onto the filter membrane of the funnel, and when there is no droplet in the funnel, the fungal mycelium on the wire mesh is collected, and the liquid culture of fungal bacteria is completed. Collection of filaments. The steel wire mesh in this embodiment can be reused after being washed with sterile water, which is energy-saving and environmentally friendly, and will not cause cross-contamination of mycelium of different samples.

Example 2

The invention discloses a method for collecting fungal mycelium in liquid culture, which adopts a vacuum suction filtration method, and the filter membrane adopts a 150-250-mesh steel wire mesh. The suction filtration system includes a vacuum pump, a suction filtration bottle connected with the vacuum pump, and a funnel installed on the suction filtration bottle, and a filter membrane made of 150-250 mesh steel mesh is placed in the funnel. When collecting fungal mycelium, pour the ready-to-use fungal fermentation liquid into the filter funnel, turn on the vacuum pump for vacuum filtration, and collect the fungal mycelium on the wire mesh and the suction filtration system separately until there is no droplet in the funnel. The fungal fermentation liquid in the suction filter bottle gets final product.

The water content of fungal mycelium collected by the method of this embodiment can be as low as 80% ± 5%, which is simple and fast. After the steel mesh is washed with sterile water, it can be reused, energy-saving and environmentally friendly, and will not cause crossing of mycelia from different samples. pollute. It has a wide range of applications, and is suitable for viscous fermented liquid (such as Cordyceps sinensis, Cordyceps militaris) or relatively clear fermented liquid (such as: oyster mushroom, ganoderma, etc.).

Example 3

A method for collecting fungal mycelium in liquid culture, the steps are as follows:

1) Cut the 150-mesh steel wire mesh into a circle with the same caliber as the Buchner funnel;

2) Place the circular steel wire mesh in the Buchner funnel and press it to form;

3) Install the vacuum filtration system, including connecting the vacuum pump to the filtration bottle, and placing the Buchner funnel in the mouth of the filtration bottle;

4) Pour the fungal fermentation broth to be collected into the Buchner funnel, and open the suction filter;

5) When there is no liquid drop from the tail of the Buchner funnel, collect the fungal mycelium on the wire mesh and the fungal fermentation broth in the suction filter bottle respectively to complete the collection of the fungal mycelium.

Example 4

A kind of collection method (vacuum suction filtration method) of liquid culture shiitake mushroom mycelia, the steps are as follows:

1) Cut the 200-mesh steel wire mesh into a circle with the same caliber as the Buchner funnel;

2) Place the circular steel wire mesh in the Buchner funnel and press it to form;

3) Install the vacuum filtration system, including connecting the vacuum pump to the filtration bottle, and placing the Buchner funnel in the mouth of the filtration bottle;

4) Pour the mushroom fermentation broth to be collected into the Buchner funnel, and open the suction filter;

5) When there is no more droplet at the end of the Buchner funnel, collect the mushroom mycelium on the wire mesh and the mushroom fermentation liquid in the suction filter bottle respectively to complete the collection of the mushroom mycelium (as the experimental group).

Similarly, the above method for collecting shiitake mycelium was used to collect the mycelium of Pleurotus ostreatus and the mycelium of black fungus, which were respectively used as experimental groups.

In addition, the above-mentioned vacuum filtration method was also adopted, but filter paper was used instead of steel wire mesh as a filter membrane, and mushroom mycelium, oyster mushroom mycelium and black fungus mycelium were collected respectively, which were respectively used as control group 1.

In addition, the mycelium of shiitake mushroom, the mycelium of oyster mushroom and the mycelium of black fungus were collected respectively by high-speed centrifugation at 12000 rpm, which were used as control group 2 respectively.

Weigh the wet weights of the collected three kinds of mycelia (three groups: experimental group, control group 1 and control group 2), dry them at 80°C, and weigh their dry weights again, and finally obtain the collected three kinds of mycelium Wet weight and dry weight of the three groups, and the moisture content is obtained by calculation (moisture content=(wet weight-dry weight)/wet weight×100%), as shown in Table 1.

Table 1: Moisture content table of three groups of three kinds of mycelia

Control group 1: Mycelium collected by high-speed centrifugation; Control group 2: Mycelium obtained by vacuum filtration using filter paper as a filter membrane; Experimental group: Mycelium obtained by vacuum filtration using steel mesh as a filter membrane .

a stands for very significant difference compared with control group 1 (P<0.01); b stands for significant difference compared with control group 1 (P<0.05); c stands for both compared with control group 1 and control group 2 Very significant difference.

It can be seen from Table 1 that for the three kinds of fungal mycelia treated in three ways, the water content of the mycelia obtained by the high-speed centrifugation method in control group 1 is the highest, while the water content of the experimental group is the lowest. Wherein, for the mycelium of different fungal strains, the reduction rate of the water content of the obtained mycelium is different when using steel wire mesh as the filter membrane and using filter paper as the filter membrane. The reduction rate of the water content of the silk is the smallest, and the reduction rate of the water content of the black fungus mycelium is the largest, that is, the effect is the most significant.

According to the analysis, the control group 1 adopts high-speed centrifugation to collect mycelia. For the viscous fermentation broth, the mycelium is difficult to settle. For the large mycelium balls, the liquid contained in the mycelium balls cannot be removed by centrifugation. , so the mycelium collected by this method has a high moisture content, takes a long time to dry, and is more likely to be polluted during the drying process. At the same time, this method also requires the purchase of centrifuges, rotors of different specifications, and centrifuge tubes of different specifications. The cost is high, and high-speed centrifugation takes a long time and is difficult to operate. When the weight of the sample is not well balanced, high-speed centrifugation is easy to damage the rotor. Centrifuge tubes may even cause accidents that may cause injuries.

Control group 2 uses filter paper as a filter membrane to collect mycelium by vacuum filtration. For clearer fermentation broth, the suction filtration effect is better, and the mycelium ball can be crushed with a metal medicine spoon to release the fermentation broth inside. However, the micropores of the filter paper are small and easy to clog, and the filter paper needs to be replaced to cause waste; for the viscous fermentation liquid, the filter paper will be blocked quickly, and frequent replacement will cause sample loss and waste. The obtained mycelium is pasty, has a high water content, takes a long time to dry, and is easy to be polluted during the drying process.

In the experimental group, steel wire mesh was used as a filter membrane for vacuum filtration to collect mycelium. Because the steel mesh has a large pore size, it is not easy to cause blockage, and it is not easy to cause mycelium to leak out. Therefore, the separation effect of mycelium and fermentation broth is good. The collected mycelium has low water content and a wide range of applications. Fungal fermentation with different characteristics can collect mycelium with a lower water content. Ganoderma lucidum mycelium, Zizhi mycelium with 79.6% moisture content, Hericium erinaceus mycelium with 81.5% moisture content, Oddya mycelium with 83.6% moisture content, Flammulina velutipes mycelium with 83% moisture content, 80.1 % water content of blood mushroom mycelium, 81.7% of water content of the mycelia of Jasmine mushroom. In addition, the price of 200-mesh steel wire mesh (304 food-grade steel) per square meter is about 40 yuan, which can be made into 150-170 pieces of steel wire mesh for filtration, which is low in cost and can be reused repeatedly.

The above description is only a preferred specific embodiment of the present invention, but the protection scope of the present invention is not limited thereto. Any equivalent replacement or change of the scheme and its concepts shall fall within the protection scope of the present invention.

Claims (8)

1. a collection method of liquid culture fungal mycelium, is characterized in that, adopts filtration method, with steel wire mesh as filter membrane. 2. the collection method of a kind of liquid culture fungal mycelium as claimed in claim 1, is characterized in that, described steel wire mesh is 150-250 mesh steel wire mesh. 3. the collection method of a kind of liquid culture fungal mycelia as claimed in claim 2, is characterized in that, described steel wire mesh is 200 mesh steel wire meshes, and described fungus is shiitake mushroom or oyster mushroom or black fungus. 4. A method for collecting liquid cultured fungal mycelia according to any one of claims 1 to 3, wherein the filtration method is a vacuum filtration method. 5. the collection method of a kind of liquid culture fungal mycelium as claimed in claim 4, is characterized in that, comprises the filter membrane that 150-250 purpose steel wire mesh is made as funnel, fungal fermented liquid is poured into suction filtration system In the funnel, start suction filtration, and when there is no liquid drop in the funnel, collect the fungal mycelium on the wire mesh. 6. the collection method of a kind of liquid culture fungal mycelium as claimed in claim 5, is characterized in that, described suction filtration system comprises vacuum pump, the suction filtration bottle that is connected with vacuum pump, and the funnel that is installed on the suction filtration bottle . 7. The collection method of a kind of liquid culture fungal mycelium as claimed in claim 6, is characterized in that, described funnel is Buchner's funnel. 8. the collection method of a kind of liquid culture fungal mycelium as claimed in claim 5, is characterized in that, described 150-250 purpose steel wire net is made as the filter membrane of funnel and comprises that 150-250 purpose steel wire net is cut into A circle with the same caliber as the funnel, then place the steel mesh in the funnel, press and fit it into shape.