JPS5840463B2 - High bacterial cell concentration culture method for yeast - Google Patents

Description

DETAILED DESCRIPTION OF THE INVENTION In the yeast aerated culture method of the present invention, the culture solution is continuously or intermittently extracted from the system, and the yeast cells are separated using a cell separator or separated and washed with water. Only the yeast cells are returned to the culture tank and cultured until the final cell concentration in the batch culture method is 6% or more, and the cell concentration in the culture tank is 6% or more in the case of the continuous culture method. This article relates to a method for culturing yeast at high bacterial cell concentration.

Conventionally, methods for increasing the bacterial cell concentration in aerated yeast culture have mainly focused on methods for supplying nutrients and methods for increasing the amount of oxygen supplied.

For example, regarding the latter, development of a fermenter with a high oxygen supply rate, a method using oxygen (Japanese Patent Publication No. 51-9833), etc. can be mentioned.

However, it is impossible to significantly increase the yeast cell concentration using only such measures.

That is, in general, the factors that stop the growth of bacterial cells in yeast aeration culture are considered to be as follows.

■ Lack of main carbon sources, inorganic salts, and other nutrient sources ■ Insufficient oxygen supply ■ Inhibition due to accumulation of bacterial metabolites ■ Inhibition due to accumulation of salts, etc. Therefore, it is not possible to solve only ■ and ■ of the above items ■ Due to items 1 and 2, culture is inhibited and the bacterial cell concentration cannot be increased significantly.

Among the above four factors, the present inventors focused on solutions for ■ and ■, and as a result of various studies, in order to solve problems We have discovered that the most effective method is to extract the yeast cells, separate them, or separate them and wash them with water, and then culture them while returning only the yeast cells to the culture tank.

To explain (2) in more detail, the substances that accumulate in the culture solution as metabolites vary depending on the type of yeast, the type of main carbon source, culture conditions, etc.

For example, baker's yeast (Sacchar) uses sugar as the main carbon source.
When yeast (omyces cerevisiae) is cultured, alcohols, organic acids, and other metabolites are metabolized in the culture solution, and when these accumulate, the growth rate of the yeast is inhibited and the quality is also reduced.

The same holds true for cultivation with other yeast genera and other primary carbon sources.

Regarding (2), for example, when culturing using a molasses solution as the main carbon source, the molasses solution contains many salts and non-assimilable sugars that cannot be assimilated by yeast, and these are contained in the culture solution. If it accumulates, it will have an adverse effect on proliferation, etc.

The method of the present invention is an extremely advantageous method that can simultaneously solve the inhibition caused by (1) and (2).

Therefore, the conventional Invention Patent Publication No. 51-
It is clearly different from 9833.

In order to solve the problem (2) in the method of the present invention, a method of introducing oxygen during aeration to aerate highly concentrated oxygen-containing air, or a method of pressurizing the fermenter may be used, or a method of pressurizing the fermenter may be used. Depending on the method used, it is possible to grow yeast cells to an extremely high concentration.

As described above, the method of the present invention has great effects on industrial activities such as rationalization of manufacturing equipment and wastewater treatment equipment.

In particular, the present inventors have developed the idea that if the yeast cells are cultured at a much higher concentration than the yeast cell concentration previously reported, the growth of so-called miscellaneous bacteria, which are cells other than the target yeast cells, will be weakened. Based on this, the present invention was completed after intensive research.

Generally, if bacteria are mixed into the culture solution, the quality of the product will be affected or the bacteria will become predominant.

Furthermore, in the continuous culture process, it is extremely important in the fermentation industry to consider preventing the growth of various bacteria since the operating time of the continuous culture is limited.

In order to confirm the fact that it prevents the growth of bacteria,
As a result of various studies, the present inventors obtained the following experimental results regarding yeast culture.

That is, as the concentration of the desired yeast cells increases, the proliferation of miscellaneous bacteria is weakened, and the yeast concentration decreases to 6% (dry weight,
It has been found that when the temperature exceeds (the same applies hereinafter), the proliferation of various bacteria is extremely weakened.

As a result of continuous single-tank culture of various yeast genera, the general trend of changes in the number of bacteria over time was measured using a microscope, and the trends are shown in Figure 1.

The horizontal axis in FIG. 1 is the culture time and the vertical axis is the number of bacteria.

FIG. 1 shows that when the yeast cell concentration is set to 6% or more, preferably 8% or more, the effect of preventing miscellaneous bacteria is extremely large.

Although the upper limit of the yeast cell concentration cannot be absolutely defined depending on the type of bacteria, especially the size of the bacteria, it is approximately 20%.

In addition, the method of the present invention was applied to the batch culture method, and the yeast cell concentration was reduced to 6.
%, the culture time is generally longer than when the culture is completed at a low concentration.

For this reason, if the initial bacterial cell concentration is lowered, the time required to culture yeast to a concentration that prevents harmful bacteria will increase, and the harmful bacteria will proliferate during that time, so that the prevention effect will be lost.

Therefore, it is generally necessary to start culturing at an initial bacterial cell concentration of 0.11% or more, preferably 0.5% or more.

As described above, the basic method of the present invention is to increase the number of yeast cells to a large extent in order to suppress bacterial contamination, and the means for this purpose is to eliminate the aforementioned factors that stop the growth of yeast cells. .

Therefore, the method of the present invention is advantageous in terms of product quality, and the ability to extend the operating time of continuous culture is a great advantage in industrial activities.

Examples of yeast cells that can be applied to the method of the present invention include Saccharomyces genus, Candida genus, Rhodotorula spp.
genus orula, Torulopsis
), Hansenula , P 1chia , Debaromyces
yomyces), Chizosa'calomyces (S ch
The genus Izosaccharomyces and the genus Trichosporon are applicable to the genus Glucose and molasses, etc., normal paraffin hydrocarbons, alcohols such as ethanol and methanol, fatty acids such as acetic acid, soybean oil and fish oil. Any of the above-mentioned yeasts may be used as long as they can assimilate fats and oils, or a mixture of two or more types may be used.

The main carbon sources applicable to the method of the present invention include sugars, hydrocarbons, alcohols, fatty acids, and fats and oils, and a mixture of two or more of these may also be used.

Further, as is clear from the above-mentioned principle, the method of the present invention can be applied to all types of culture tanks, such as culture tanks equipped with a stirrer and tower-mounted culture tanks.

The process of the present invention will be explained with reference to the drawings.

FIG. 2 shows a typical embodiment of the present invention, in which 1 is an air supply pipe, 2 is a main carbon source and nutrient source supply pipe,
By guiding the culture solution from the culture tank 3 through the tube 4 to the bacterial cell separator 5 and returning the yeast cells to the culture tank 3 through the tube 8, the inhibitory substances accumulated in the culture tank are removed from the bacterial cell separator 5. This can be removed from 5.

Regarding the air supply pipe 1, it is effective to introduce oxygen and aerate highly concentrated oxygen-containing air when the oxygen supply capacity of the fermenter is insufficient.

However, in this case, in order to prevent the negative effects of so-called oxygen,
It is important to control and culture the dissolved oxygen concentration within the optimal range determined by the bacterial species, main carbon source, etc.

For this purpose, it is preferable to ventilate air containing high concentration of oxygen, usually 30% or more, preferably 30 to 60%.
Air and oxygen may be vented into the fermenter from separate locations to provide the same function as aeration with highly oxygenated air.

It is also effective to pressurize the culture tank 3 for the purpose of increasing the amount of oxygen supplied.

In this case, applying a pressure of 0.5 to 5.0 kg and 104 G will normally provide sufficient oxygen supply, but higher pressure is never advantageous due to the pressure resistance of the fermenter, the equipment cost, and the equipment cost of the aeration compressor. isn't it.

As for the bacterial cell separator 5, it is easy to operate and most effective to use a centrifugal separator that is generally used for bacterial cell separation.

When a membrane separator such as a membrane filter is used to separate yeast, as the bacterial cell concentration increases, the permeation rate of the membrane decreases significantly, so it is necessary to perform separation while diluting with washing water.

The amount of culture fluid withdrawn and the amount of water for washing are determined by the bacterial species, the type of main carbon source, and the degree of accumulation of inhibitors, but in general, for both batch and continuous culture methods, the amount of fluid withdrawn is determined by the amount of water removed from the fermenter. 0.3 to 4 times the total amount of the main carbon source and other feed liquids (hereinafter referred to as the supply liquid amount) to be equal to the amount of separated liquid separated from the bacterial cell separator 5. Just take it out.

Generally, an amount of washing water up to twice the amount of separation liquid is effective, but as described above, when using a membrane separator, about 2 to 5 times the amount of washing water is required.

In this case, the liquid extraction method may be continuous or intermittent (
For example, the sample may be extracted every hour), and the selection may be made as appropriate depending on the type of bacteria, main carbon source, continuous culture method, batch culture method, etc.

Furthermore, for the purpose of preventing bacterial contamination, the washing water may be sterile water that has passed through a sterilizing filter, or sterile water that has been sterilized by heat sterilization or the like.

The cooler 9 is used to remove the heat generated by the bacterial cells in the culture tank, and may be a plate-type or tube-type heat exchanger used for heat exchangers. In some cases, it may not be necessary to use it.

When applied to a continuous culture method, the yeast product may be extracted from the front of the separator 5 using the extraction tube 10, or may be extracted after separation.

Furthermore, before the yeast cells separated by the separator 5 are returned to the culture tank 3, the main carbon source and nutrient source may be added through the additional supply pipe 2a, or air may be supplied through the additional supply pipe 1a. A method of preventing deterioration of yeast cell activation may also be used.

Next, the method of the present invention can of course be applied to a multistage continuous culture method.
As an example, a part of the application example in the case of a two-stage type is shown in Figures 3 and 4.
As shown in the figure.

In these cases as well, of course, the main carbon source and nutrient source may be added when the separated yeast cells are returned to the culture tank as shown in FIG. 1, and air may be supplied.

Furthermore, a cooler 9 may be incorporated.

Next, the present invention will be explained in more detail by describing examples.

Example 1 and Comparative Example 1 Bacteria used: Carbon source and nutrient source 40% molasses solution (produced from 1 ml) was used as the main carbon source,
In addition, ammonium sulfate and urea were used as the nitrogen source, and phosphoric acid was used as the phosphorus source.

Culture conditions: Using a 301-volume jar fermentor, temperature: 33°C, P
Control with 10% ammonia water so that H4.5 to 5.0.

Aeration and stirring conditions, etc. Aeration amount is 20 N, /: 7 ml yr, number of stirring is 40
It was set to 0 ppm.

Aeration was controlled by increasing the oxygen partial pressure using an oxygen cylinder so that the dissolved oxygen concentration in the culture solution was always 2 to 7 ppm.

In this case, the ventilation amount is 20 N including air and oxygen.
l: /min.

Cultivation method 400ml of 46% urea solution, 80g of ammonium sulfate, and 60f of 85% phosphoric acid solution were added as nutrients before starting the culture. dry weight measurement method (measured at 110°C for 7 hours).

The main carbon source was added so that the ethanol concentration in the culture solution was 500 to 3000 ppm (measured by gas chromatography) in each case, in order to supply just the right amount according to the growth of the bacteria. .

The culture time was 16 hours in all cases, and batch culture was performed.

In this example, after 8 hours had elapsed from the start of culture, the culture solution was extracted from the culture tank, the bacterial cells were separated within 20 minutes using a centrifuge (Westfalia model LWA205), and only the bacterial cells were transferred to the culture tank. The cells were cultured while being returned to .

This separation operation is performed at hourly intervals, and the operation is performed so that the volume of the main carbon source liquid added during that hour is approximately equal to the volume of the separated liquid that does not contain bacterial cells separated by the centrifuge. I went to the end.

In Comparative Example 1, culture was performed under the same conditions except that bacterial cell isolation was not performed.

Culture results The final yeast amount obtained is shown in the table below.

The total amount of yeast was determined by measuring the concentration using a dry weight measurement method (110° C., 7 hours).

The yield was expressed as the percentage of dried bacterial cells produced relative to the amount of sugar added.

Example 2 and Comparative Example 2 Bacterial strains used Same main carbon source and nutrient source as Example 1 and Comparative Example 1 Same culture conditions as Example 1 and Comparative Example 1 Same aeration stirring conditions, etc. Aeration amount as Example 1 and Comparative Example 1 Culture was carried out under conditions of 2 ONl/min, stirring speed of 700 rpm, and pressurization of 1 kg/cstG in a jar fermentor.

Cultivation Method The culture was carried out in the same manner as in Example 1, and in Comparative Example 2, the culture was carried out under the same conditions except that the bacterial cells were not isolated.

Culture results The measurement method and calculation method are the same as in Example 1 and Comparative Example 1.

Example 3 and Comparative Example 3 Bacterial strain used Candida utilis IAM 421
5 Main carbon source and nutrient source 30% molasses liquid (produced in the Philippines) was used as the main carbon source, other culture conditions were the same as in Example 1 and Comparative Example 1, using a 301-volume jar fermenter, temperature 28°C, P
Single-tank continuous culture operation was performed from H4.5 to H50.

Aeration and stirring conditions, etc. Same culture method as Example 1 and Comparative Example 1 A feed solution containing 0.5% urea, 0.2% ammonium sulfate, and 0.2% phosphoric acid added to a 30% molasses solution. 3. Drop culture conditions: Using a 301 volume jar fermenter, feed continuously at the temperature * J/H, and reduce the liquid volume in the jar fermenter to 20
I controlled it to 7.

Separation and water washing by the method of the present invention were carried out using the same centrifugal separator as in Example 1, with continuous extraction using a 317H pump.

In this case, a separation operation was carried out while the washing water was continuously mixed with the culture solution from which 3 J/H was extracted in a centrifuge, and only the separated bacterial cells were returned to the jar fermentor.

In Comparative Example 3, culture was performed under the same conditions except that bacterial cell isolation was not performed.

In both the culture results of Comparative Example 3 and the present invention, after confirming that the continuous operation has reached a stable, so-called steady state, the concentration of bacterial cells was measured by dry weight measurement, and the production rate of bacterial cells was determined. and the yield was determined.

The yield was the same as the method determined from the culture results of Example 1 and Comparative Example 1.

Example 4 and Comparative Example 4 Strain used Same as Example 3 and Comparative Example 3 Main carbon and nutrient sources The main carbon source is 30% ethanol and other nutritional sources are as shown in the table below. Batch culture was performed at 33°C and pH 5.0.

Aeration and stirring conditions, etc. Same as Example 1 and Comparative Example 1.

Cultivation method: The above-mentioned nutrient source was added to an initial volume of 101 ml of the initial solution, and the initial bacterial concentration was 1% (dry weight method).

The main carbon source was added so that the ethanol concentration in the culture solution (measured by gas chromatography) was always 0.2 to 0.5%.

The culture time was 18 hr in each case, and the separation operation was performed in the same manner as in Example 1 and Comparative Example 1.

In Comparative Example 4, culture was performed under the same conditions except that no bacterial cell isolation operation was performed.

Culture results The final yeast amount obtained is shown in the table below.

The measurement method and calculation method are the same as in Example 1 and Comparative Example 1.

HelJI! IV J J /)t+'-' j-U%J
Pichia mogii IFOO607 was used as the carbon source and nutrient source. Soybean oil was used as the main carbon source, and other nutrient sources were the same as in Example 4 and Comparative Example 4.

Musical conditions Same as Example 4 and Comparative Example 4.

Sound Ceremony Method As mentioned above, the nutrient source was added to the initial solution volume of 15 J to achieve the above concentration, and the initial bacterial concentration was started at 1%.

Soybean oil, the main carbon source, was added so that the concentration in the culture solution was always about 0.2%.

The culture time was 18 hours, and the separation operation was performed 1 time after the start of culture.
At 0 hr, 81 was extracted, mixed with washing water 81, and separated within 1 hour using the same centrifugal separator as in Example 1 and Comparative Example 1, and the 81 separated liquid was removed.

Thereafter, the culture was continued for a total of 18 hours.

In Comparative Example 5, culture was performed under the same conditions except that bacterial cell isolation was not performed.

Culture results The final yeast amount obtained is shown in the table below.

The measurement method and calculation method are the same as in Example 2.

[Brief explanation of drawings]

FIG. 1 is a graph showing the relationship between yeast concentration and the number of miscellaneous bacteria, and FIGS. 2 to 4 are flow sheets showing embodiments of the present invention. 1 is an air supply pipe, 1a is an additional air supply pipe, 2 is a carbon source and nutrient source supply pipe, 2a is an additional carbon source and nutrient source supply pipe, 3 is a culture tank, 4 is a culture solution extraction pipe, 5 is a bacterial cell separator, 6 is a washing water supply pipe, 7 is a tube for extracting the separated liquid containing inhibitors separated by the bacterial cell separator, 8 is a tube for removing yeast cells separated by the bacterial cell separator, and 9 is a pipe for extracting yeast cells separated by the bacterial cell separator. A cooler for cooling the yeast cells, and 10 a liquid extraction tube for continuous culture.

Claims (1)

[Claims] 1. In the yeast aerated culture method, the culture solution is continuously or intermittently extracted from the system, and the yeast cells are separated using a cell separator or separated and washed with water, and then the yeast cells are separated. Culture is carried out while returning only the cells to the culture tank, and in the case of batch culture method, the final cell concentration is 6%, and in the case of continuous culture method, the cell concentration in the culture tank is 6%.
A method for culturing yeast at a high bacterial cell concentration in the range of 20% (dry weight). The culture method according to claim 1, wherein the final bacterial cell concentration in the case of two-batch culture or the bacterial cell concentration in the culture tank in the case of continuous culture is 8% or more. The culture method according to claim 1, wherein the initial bacterial cell concentration is 0.1% or more when cultured in 3 batches. 4. The culture method according to claim 3, wherein the initial bacterial cell concentration is 0.5% or more. 5. The culture method according to claim 1, wherein highly concentrated oxygen-containing air containing an oxygen concentration of 30% or more is used for aeration. 6. The culture method according to claim 1, wherein the culture is carried out by pressurizing the fermenter at a pressure of 9/c4G to 0.5 to 5.0. 7 The amount of yeast-free separated liquid separated from the bacterial cell separator (hereinafter referred to as separated liquid amount) is 0.3 to 4 times the total amount of the main carbon source and other feed liquids supplied to the fermenter. The culture method according to claim 1, wherein the liquid is extracted so that 8 Claim 1, in which a centrifugal separator is used as a bacterial cell separator, and the amount of washing water is up to twice the amount of separation liquid.
Culture method described in section. 9. The culture method according to claim 1, wherein a membrane separator is used as the bacterial cell separator, and the amount of washing water is 2 to 5 times the amount of the separation liquid. 10. The culture method according to claim 1, wherein the yeast cells are returned to the culture tank after being cooled with a heat exchanger. 11. The culture method according to claim 1, wherein the yeast cells are returned to the culture tank after adding the main carbon source and nutrient source. 12. The culture method according to claim 1, wherein the yeast cells are returned to the culture tank while being aerated. 13. The culture method according to claim 1, wherein the main carbon source is saccharide. 14. The culture method according to claim 1, wherein the main carbon source is hydrocarbons. 15. The culture method according to claim 1, wherein the main carbon source is alcohol. 16 Claim 1 in which the main carbon source is fatty acids
Culture method described in section. 17. The culture method according to claim 1, wherein the main carbon source is oil or fat. 18 Yeast is Saccharomyces
ces) genus, Candida genus,
Rhodotorula genus, Torulopsis genus, /S nzenula (
Hansenula) genus, Pichia (P 1chia)
Genus, Debaryomyces, Schizosaccharo
myces) genus, Trichosporon (Trichos)
2. The culture method according to claim 1, wherein the culturing method is selected from the group consisting of the genus Poron.