JPS6014399Y2 - Simple pure culture device for microorganisms - Google Patents

Description

[Detailed explanation of the idea] The present invention relates to a simple pure culture device for microorganisms.

Pure culture of microorganisms is usually carried out in batch-type, robust culture equipment.

However, this culture method requires sterilization of the equipment, injection of culture medium,
Inoculation, observation of the state of growth of microorganisms, culturing, exchange or adjustment of the culture solution, removal of microorganisms, etc. must be carried out under very strict and controlled conditions.

Moreover, if heat sterilization is performed with the aim of maintaining the purity of the microorganisms to be cultured, the quality of the culture solution, especially its heat-unstable components, will change, which will impede good culture.

On the other hand, aseptic filtration of a culture solution not only requires more trouble than heat sterilization, but also has drawbacks such as the filtration membrane being easily clogged.

Furthermore, since there is a limit to the size of the culture device, there is also a limit to the amount of microorganisms that can grow, and it has been extremely difficult to obtain a large amount of microorganisms by pure culture.

The purpose of the present invention is to provide a simple pure culturing device for microorganisms that solves the above-mentioned problems.

The culture device of the present invention will be explained based on the drawings. The culture device of the present invention comprises a tubular container 1 formed of a semipermeable membrane and a stopper 2 for closing the opening of the container.

In the present invention, the tubular container 1 is formed by a semipermeable membrane.

There are no restrictions on the material of the semipermeable membrane used in this step, but it is preferably heat resistant for convenience of culture operations.

For example, Visk manufactured by Visking Co., Ltd.
ing Celulose Tubing and the like.

Depending on the type of microorganism to be cultured, it cannot be said that there is no possibility of destroying the cellulose tube, but such microorganisms are actually extremely rare, so this does not impede the practicality of the present invention.

Generally, carbon sources, nitrogen sources, inorganic components, etc. required by microorganisms have a molecular weight of 1000 or less and can permeate through a semipermeable membrane.

On the other hand, microorganisms, even microscopic bacteria, are unable to pass through semipermeable membranes.

Therefore, the pore size of the semipermeable membrane may be any size as long as it prevents passage of bacteria and viruses.

Cultivation of microorganisms has troublesome problems such as foaming and staining of the cultured microorganisms, which can occur during the brewing of beer, sake, and wine, but if they are separated by a semipermeable membrane, these problems within the membrane can be greatly reduced. can be reduced.

There are no restrictions on the diameter, length, etc. of the tubular container, and they may be appropriately determined in consideration of the intended use.

Practically, it is appropriate that the internal capacity is from several rrLl to several liters.

Note that it is preferable that the content of this container is larger than the amount of the microorganism suspension to provide an empty space.

The tubular container can be produced by various methods, but as a simple method, it can be produced by tying one end of a semipermeable membrane tube with a string or by bonding it with an adhesive.

Next, the stopper 2 may be of any type as long as it can close the opening of the tubular container 1, but preferably a porous one made of a resin foam such as foamed silicone resin is used.

The porous resin foam stopper regulates the internal pressure of the container by dissipating gases produced by fermentation, and allows gas exchange with the outside to regulate the pressure within the container.

Moreover, it is possible to inoculate microorganisms and take out samples from containers through the needle of the syringe.

Additionally, this stopper can be heat sterilized.

In addition, in the case of microbial culture that does not involve gas rawhide, instead of using a stopper, the opening can be sealed aseptically using a rubber stopper, the above-mentioned string, adhesive, or the like.

When performing pure culture of microorganisms using the culture device of the present invention, not only can the exact same conditions (culture solution composition, temperature, ventilation, etc.) be applied as in culture methods using conventional batch culture devices, but also Furthermore, it is also possible to adopt the following distinctive culture conditions.

In addition, when the culture device tends to float, a weight can be attached to the tubular container and most of the container can be immersed in the liquid to be in a bathing state.

■ The concentration of the culture solution can be increased.

For example, in the cultivation of microorganisms in which a glucose concentration of 5% is considered appropriate in the Batsuji culture method, according to the present invention, the glucose concentration of the liquid outside the semipermeable membrane may be set to 8%.

■ Culture can be carried out without any problem even if the culture solution contains polymeric substances, sticky substances that are difficult to filter, or substances that should not come into contact with the microorganisms (e.g., enzymes, viruses, etc. that act on the microorganisms being cultured). , preparation of culture solution and fermentation management are easy.

■ No infection occurs even if different types of bacteria are present in the culture solution.

Therefore, normal culture is possible without sterilizing the culture solution.

In addition, deterioration of the culture solution due to sterilization can be avoided.

Although the basic shape of the culture device of the present invention is illustrated in FIG. 1, it goes without saying that it can be modified as appropriate depending on the purpose of use.

A specific example is shown below. ■ When culturing in a fermentation liquid that is highly concentrated. For example, in beer fermentation, bubbles can reach a height of several pupil heights. In such cases, the stopper of the culture device of this invention may If buried, handling becomes inconvenient, so the tubular container can be extended with a suitable tube from the surface of the culture medium to a position out of reach of bubbles.

At this time, the extension tube may be connected to the outside of the tubular container and the stopper may remain in the same position, or the stopper may be moved to the tip of the extension tube.

As mentioned above, the bubble height in the culture vessel is significantly lowered by this method, and there is no need to provide an extra empty space.

(2) When culturing in a flowing waterway For example, when culturing photosynthetic bacteria, a long and thin semipermeable membrane tube is used as the tubular container of the present invention so that it lies horizontally on the liquid surface along the flow.

It is also desirable to connect the stopper to a fixed object above the liquid surface.

■ When culturing in semi-solid matter For example, when culturing in sake moromi or red grape sake liquor,
The outside of the culture device of the present invention is protected by surrounding it with a suitable net.

This allows the characteristics of the semipermeable membrane to be exhibited.

A comparison of the effectiveness of the culture device of the present invention with a conventional batch culture device showed that the purity of the cultured microorganisms was maintained at the same level, and the concentration of microorganisms was several times to W times higher in the same container, and even during the growth phase. It was confirmed that suitable bacteria could be retained for immediate mass culture.

The device of the present invention also has the advantage of being inexpensive to manufacture.

Next, an example of use of the culture device of the present invention will be shown.

Usage example l As a semipermeable membrane tubular container, Visking's cellulose tube for dialysis is used with a flat surface of 33 mm and an internal capacity of 100 rIL.
A container adjusted to be L in size was used, and distilled water 707FLL was poured into the container, a plug of porous silicone foam resin (manufactured by Shin-Etsu Chemical Co., Ltd.) was attached, and the container was heat sterilized in an autoclave.

Next, 0.37 FL of a suspension of beer brewing yeast (Saccharomyces carlsbergensis IAM-4788)
L was injected into the container through the stopper using a syringe.

The culture device of the present invention injected with yeast is 200WLL.
The samples were immersed in a graduated cylinder containing 160 iL of wort, kept in a constant temperature room at 10°C, and the total volume, dead cell rate, and number of bacteria were examined every week.

The culture apparatus of the present invention was moved to a graduated cylinder filled with new wort every week.

The number of dead cells was measured by methylene blue staining.
The number of bacteria was measured by the achinadione addition plate counting method.

On the other hand, as a control for comparison, a 100ml Erlenmeyer flask was fitted with a cotton stopper, and after dry heat sterilization, 70rrLL of wort was added and heat sterilized by autoclaving. The cells were kept in a room, and the total number of fractions, dead cell rate, and number of bacteria were examined every week.

As a result, as shown in Figure 2, the total amount of yeast and the number of bacteria increased significantly from the 4th week when using the culture device of this invention, but no increase was observed after the 1st week in the case of the control. I couldn't.

In addition, the dead cell rate gradually increases as the week progresses, but at the 7th week, it is approximately 6.
%, and the control was about 8%.

The number of bacteria was O in both cases, confirming that pure culture was performed.

Usage example 2 Add 70rr of distilled water to the same culture device as used in usage example 1.
LL was placed in the container, a cap made of porous foamed silicone resin was attached, and the container was heat sterilized in an autoclave, and then the same yeast was injected into the container in the same manner as in Use Example 1.

Soak this in a container containing 100rnL of wort for 15 minutes.
Culture was carried out at ℃ for 10 days.

On the other hand, as a control for comparison, a cotton swab was attached to a 100 rnL Erlenmeyer flask, and after dry heat sterilization, 70 rnL of wort was placed in an autoclave and heat sterilized, and the same amount of the same yeast was inoculated and cultured under the same conditions as above. .

In order to examine the stains or adhering substances on the cell surface of the yeast cells obtained in this way, the culture solution was centrifuged to collect the bacteria.
The number of cells per y of yeast that had been lightly washed with water was determined.

The results are shown in Table 1. Table 1: Number of yeast cells (X106 cells/g) 12345 Average of this invention Nyoru culture 5Cf30423036003840
=49904344 Culture by symmetry 32303770
321034-7035103438 As is clear from the above results, it was confirmed that the yeast obtained by culturing using the apparatus of the present invention had a large number of cells per y and had few surface deposits.

This indicates that there is less adhesion and contamination of macromolecular proteins and polysaccharides that are separated by the semipermeable membrane.

Furthermore, as a result of microscopic examination, the sizes of the yeast obtained in both cultures were the same.

Usage Example 3 25 rILL of distilled water was placed in a semipermeable membrane tubular container with a content of 30 rILL, a silicone resin column was attached, and the container was sterilized in an autoclave.

After cooling, culture solution of lactic acid bacteria (Lactobacillus delbrueckii IAM-1149) (3,000,0
OOffil/mj) 0.3rrcl was inoculated into the container through the stopper using a syringe.

The culture device injected with lactic acid bacteria was immersed in a 10W graduated cylinder filled with corn/sugar-barley-phosphoantine culture solution with a sugar concentration of 15%, and then placed in a 48°C incubator for 6 days. did.

The culture solution was replaced with a new one every day.

For comparison, the same lactic acid bacteria were inoculated into the above culture solution that had been sterilized by adding an excessive amount of lime carbonate, and batch culture was performed under the same conditions.

When the lactic acid bacteria concentration was examined after 6 days, it was 15.000.00 m/it using the device of the present invention.
The control was 2.500. It was OOso/rlLL.

Note that when the device of the present invention is used, carbonate lime for neutralization is not required.

[Brief explanation of drawings]

FIG. 1 is a sketch of an embodiment of the culture apparatus of the present invention, and FIG. 2 is a graph showing the relationship between the number of culture days and the weight of microorganisms.

Claims (1)

[Claims for Utility Model Registration] 1. A simple pure culture device for microorganisms comprising a tubular container formed of a semipermeable membrane and a stopper or equivalent material for closing the opening of the container. 2. The device according to claim 1, wherein the stopper is made of resin foam. 3. The device according to claim 1, wherein the stopper is made of foamed silicone resin.