JPH0440888A - Production of fungus containing polymerized selenium - Google Patents

Abstract

PURPOSE:To obtain the title fungi at a high proliferation rate by dispersing fungi produced by incubating specific microorganisms in an aqueous solution containing an organic compound as substrate and a water-soluble selenium compound. CONSTITUTION:Firstly, yeast such as beer yeast, bacteria such as lactobacillus, or unicellular algae such as Chlorella is, as the case may be, incubated and proliferated. The resultant fungi collected is washed with e.g. water and prepared to a fungal concentration at a level not lower than the biological space. The fungi is then dispersed in an aqueous solution containing (A) an organic compound as substrate for polymerizing selenium (e.g. saccharide such as glucose and/or amino acid such as leucine and (B) a water-soluble selenium compound such as selenious acid, and reacted into polymerized selenium which is then accumulated in the fungi. After reaction, the fungi is collected through e.g. centrifugation and washed with water, thus giving the objective fungi at a high proliferation rate. The present fungi can be concentrated; therefore, the waste liquor therefrom can be reduced.

Description

Detailed Description of the Invention <Industrial Application Field> The present invention relates to a method for producing polymerized selenium-containing microbial cells, and the polymerized selenium-containing microbial cells obtained by the present invention can be used in animal feed,
It can be used to prevent selenium deficiency in humans or animals by adding it to feed or food.

<Prior art> Selenium is an element with an atomic number of 34 and an atomic weight of 79. Previously, it was only known as an element that it was harmful to living organisms, but in 1957, Schwarz et al. J, Am, Ches, Soc,
79:3292, 1957), it was found to be toxic and, depending on how it is used, to become an active ingredient in living organisms. Since then, the usefulness of selenium in living organisms has attracted attention, and subsequently, Sc.

11 studies have shown that selenium is effective in preventing livestock disorders caused by vitamin E deficiency, such as white muscle disease in calves, liver nephrosis in pigs, and muscle growth abnormalities (
Feedstuffs, 29, 41.20 (1957)
), and in 1969, Thompson and Scott et al. demonstrated that selenium itself is an essential element for animals (J. Nutr., 97:335, 196
9), there is no doubt that it is an essential element for humans as well.

Selenium deficiency is known to date in many species, and in Australia and New Zealand, selenium is added to mineral salts or fertilizers, or used as an adjunct to compound feed.

In addition, selenium alone or as a mixture of selenium and vitamins is widely used in selenium-deficient areas around the world, and in 1972 it was announced that selenium is a component of glutathione peroxidase.
At the end of 1974, the country's FDA approved the addition of selenium to feed, etc.
Physiological research on selenium is becoming more active.

On the other hand, there have been reports regarding organic selenium as a supply source rather than inorganic selenium, such as reports regarding combinations of selenium and amino acids and reports regarding the supply of selenium-containing microorganisms. For example, U.S. Patent No. 4,530,846 discloses a method for producing selenium yeast, and in Japan, adding inorganic selenium to feeds, foods, etc. is not approved for safety reasons, and several There have been reports regarding organic selenium. For example, Tokuko Sho 55-3
No. 6314 is a selenium-containing microbial cell that has been organicized by culturing and propagating microorganisms that can accumulate selenium in their cells in a medium to which a water-soluble inorganic selenium compound that does not significantly inhibit the growth of microorganisms has been added. Further, JP-A-57-174098 discloses a method for obtaining bioactive selenium-containing protein polysaccharides from selenium-containing yeast cells obtained by culturing in a medium supplemented with selenium compounds,
JP-A-60-180582 reports a bacterium that can accumulate L-selenone stain by culturing in a medium containing a selenium compound. In addition, methods for producing selenium-binding proteins by synthetic methods have also been reported. Furthermore, JP-A-60224451 reports a feed composition in which organic selenium-containing microbial cells obtained by culturing and propagating microorganisms in a medium to which inorganic selenium has been added are added to feed for livestock and poultry. .

<Problems to be solved by the invention> The above-mentioned methods of using organic selenium instead of inorganic selenium as a supply source involve culturing and growing microorganisms in a medium to which inorganic selenium has been added in advance. We have obtained selenium-containing bacterial cells. In this case, in order to incorporate selenium into the bacterial cells, microorganisms are allowed to grow in the presence of inorganic selenium to make the selenium organic. However, since the added selenium is inorganic selenium, the growth of microorganisms is less likely to occur than inorganic selenium. Since growth is inhibited due to toxicity, the concentration of inorganic selenium must naturally be limited to the minimum amount that does not significantly inhibit the growth of the microorganism. In addition, even if the added selenium concentration is set to the minimum level that does not significantly inhibit the growth of microorganisms, it still takes a longer time for bacterial cell growth than when culturing and propagating in a normal medium without any selenium added, resulting in a higher growth rate. Furthermore, the selenium uptake efficiency of the bacterial cells is also poor, and the yield of organic selenium-containing bacterial cells is therefore extremely low. Furthermore, because the addition of selenium is limited to a low concentration, which is the minimum amount that does not inhibit the growth of microorganisms, it is complicated to constantly measure the selenium concentration in the culture medium and add it multiple times to reach the desired concentration. Even if culture is carried out in this way, there is a limit to the concentration of bacterial cells, so the concentration of inorganic selenium that remains in the culture supernatant without being incorporated into the bacterial cells will increase, making it difficult to dispose of waste liquid. Sometimes it is necessary to remove inorganic selenium remaining in the culture supernatant.

However, since various other components are present in the culture supernatant, extremely complicated operations are required to remove inorganic selenium, leading to increased costs.

<Means for Solving the Problems> The present inventors conducted intensive research to solve the above problems, and found that organic selenium, that is, polymerized selenium, accumulates in bacterial cells. The microorganisms that can be produced are cultured and propagated appropriately in advance in a medium that does not substantially contain selenium, then the bacterial cells are collected, and if necessary, after washing with water to remove culture medium components, the bacterial cells are cultured and propagated. Conditions that do not require the use of sugars and/or amino acids, which are organic compounds that can serve as substrates for selenium polymerization, and phosphates and By dispersing it in an aqueous solution containing a water-soluble selenium compound, selenium is efficiently incorporated into the bacterial cells and combined with proteins, etc., to form polymers, surprisingly without the need for culturing or multiplying the bacterial cells. We discovered that it is possible to stably accumulate a large amount of polymerized selenium within bacterial cells, and based on the above, we established a novel method for producing polymerized selenium-containing bacterial cells.

The present invention has been completed based on the above findings, namely, by culturing microorganisms capable of accumulating polymerized selenium within the bacterial cells, the bacterial cell concentration is increased to a level higher than the biological space; A polymerized selenium-containing method characterized in that polymerized selenium is accumulated in the bacterial cells by dispersing the bacterial cells in an aqueous solution containing an organic compound that is a polymerization substrate for selenium and a water-soluble selenium compound. This is the national production method.

In the present invention, polymerized selenium accumulates in bacterial cells by dispersing them in an aqueous solution (hereinafter sometimes referred to as reaction solution) containing an organic compound and a water-soluble selenium compound that can serve as a substrate for polymerizing selenium. Estimating the process by which organic compounds such as sugars and/or amino acids that can be used as selenium polymerization substrates are first grown in a state that does not require the cultivation and proliferation of bacterial cells, preferably at a concentration higher than the biological space concentration. It is speculated that this substance becomes a substrate that generates energy within the bacterial cells when it comes into contact with the bacterial cells prepared and dispersed, and provides an energy source to activate the phenomenon of selenium in water-soluble inorganic selenium compounds being taken into the bacterial cells. be done. In other words, the microbial cells are dispersed in the reaction solution and utilize the energy generated by the action of the high-energy phosphoric acid compound synthesis mechanism, which is an intracellular reaction mechanism, based on energy substrates such as sugars and amino acids in the reaction solution. It is assumed that this reaction triggers a reaction that causes selenium to be taken into the bacterial body. High-energy phosphate compounds include adenosine diphosphate (ATP
), uranosyl triphosphate (UTP), guanosyl triphosphate (GTP), and cytosyl triphosphate (CTP), and the energy utilized in the present invention may be any of the above.

Synthesized within the bacterial body mainly using sugars and/or amino acids as substrates, selenium is first used to penetrate the bacterial cell membrane, and then the selenium that has passed through the cell membrane is taken up into the bacterial body and converted into bacterial proteins. Although it is presumed that the selenium is effectively utilized through the reaction of binding and converting it into polymeric selenium, the present invention is not limited thereto in any way.

The purpose of the production method of the present invention is to culture and proliferate the bacterial cells in a substantially selenium-free medium without adding selenium in advance, so there is no growth inhibition due to selenium during culturing, and there is no need to add selenium in advance as in the prior art. To provide a method in which the growth rate of bacterial cells is faster and the growth rate is also excellent compared to the case where cells are cultured and grown in a different medium.

In addition, in the present invention, the bacterial cells are sufficiently cultured and multiplied in a medium, and after the bacteria are collected, the bacterial cells are dispersed in the reaction solution, so that the bacterial cells can be concentrated, so the reaction system can be made smaller. To provide a method in which the amount of waste liquid after reaction can be reduced, and the waste liquid treatment can be simplified because the reaction liquid does not contain metals other than selenium.

Further, in the present invention, the cells are propagated appropriately, and after collection, the cells are washed with water to remove the culture medium components, and the cell resistance is preferably adjusted to be equal to or higher than the biological space to stop the culture and proliferation, and then the reaction solution is Since the bacterial cells are dispersed into the cells, naturally there is no need for bacterial cell proliferation during the reaction, and selenium, which inhibits bacterial cell growth, can be kept at a high concentration in the reaction solution, and selenium can be taken up into the bacterial cells. To provide a method for producing a bacterial cell containing a high concentration of polymerized selenium, which can be accumulated in a high concentration.

The present invention will be explained in detail below.

First, regarding the microbial cells to be used, we will use a wide range of microorganisms that can pass through and absorb selenium into the bacterial cells, accumulate polymerized selenium, and become microbial cells containing polymerized selenium. be able to. Such bacteria take selenium into the bacteria and combine it with proteins etc. under the conditions of an aqueous solution containing an organic compound and a water-soluble selenium compound, which are the polymerization substrates for selenium, and produce inorganic selenium through an intracellular reaction mechanism. It is presumed that it can be converted into polymeric selenium. Furthermore, since the selenium-containing microbial cells obtained by the present invention are mainly expected to be fed to animals by adding them to animal feed or feedstuffs, or to be added to foods, the microbial cells used may be Preferably, it is edible.

Examples of the above-mentioned microorganisms include yeast, bacteria, 5 molds, and unicellular green algae. Among yeasts, yeasts that are used in the production of various foods and are recognized to be safe include yeasts belonging to the genus Satucharomyces, which are known as beer yeasts, baker's yeasts, alcohol yeasts, wine yeasts, etc. Preferably Saccharomyces cerevis
7 Calomyces roxy (S.
accharomycesrouxii) and Zygosaccharomyces sp. (Zyg.

Yeasts belonging to Saccharomyces sp, ), Hansenula () J
ig, Candida spp., Torulasupora spp., Torulopsis (
Genus Torulopsis, Mycoto
A wide range of edible yeasts belonging to the genus Rula can be used in the present invention.

As for bacteria, Lactobacillus, known as lactic acid bacteria,
Lacttobacillus casei
) = Lactobacillus acidophilus (Lacto
bacillus acidphilus, etc., and Bacillus nut (Bacillus, also known as natto bacteria).
A wide range of edible bacteria such as Aspergillus niger
nigar) and Aspergillus awamori (Aspe
A wide variety of other edible molds can be used, with only a few examples including those belonging to the genus Rugilose awamori.

Chlorella vulgaris and Spirulina, which are edible unicellular green algae, can also be effectively used in the present invention.

Microbial cells capable of accumulating such polymerized selenium may be appropriately cultured and propagated in a culture medium or medium prepared in advance according to an appropriate method. For culturing, each of the target microorganisms exemplified above is cultured at a suitable culture temperature using the culture medium composition, such as carbon source, nitrogen source, inorganic, etc., or culture medium composition normally used when culturing. If necessary, the inoculum may be cultured in a preparatory medium or medium, and then cultured and propagated in each culture medium or medium as appropriate.

As for the timing to stop the culture and collect the cultured bacteria, it is sufficient to simply perform appropriate culture from the late logarithmic growth phase to the late stationary phase in the bacterial growth curve, and collect the bacteria by normal means such as centrifugation. . For example, when selenite is used as a water-soluble selenium compound, it is preferably cultured and collected as appropriate from the late logarithmic growth phase, which is the stage before selenite reductase is produced, in the bacterial growth curve to the early stationary phase. If the bacteria are grown, it is preferable that the number of proliferating bacteria is large, and the bacteria are used to prepare the subculture under conditions that do not substantially require bacterial growth, preferably at a bacterial concentration higher than the biological space. When reacted with a reaction solution containing selenic acid, even if selenium is taken up into bacterial cells, it is not affected by selenite reductase, so the production of inorganic selenium can be substantially prevented, resulting in a good polymer. Selenium-containing bacterial cells can be obtained.

For example, when yeast is used as a bacterial cell, after culturing the inoculum in YPG medium etc., it is usually grown in molasses medium at 25°C to 35°C.
C for 12 hours to 24 hours, particularly preferably 18 hours to 20 hours.
It is preferable to collect the cultured bacteria after culturing and propagating for a period of time. When lactic acid bacteria are used as the bacteria, for example, in Leuconostoc medium etc., it is usually kept at 25°C to 35°C for 15 hours to 30 hours.
Time: Particularly preferably 20 to 24 hours, the bacteria may be cultured and grown, and when mold is used, for example, YM medium is usually used at 20°C to 30°C for 30 to 50 hours, particularly preferably 35 to 50 hours. It is sufficient to collect bacteria after culturing and propagating them for 40 hours. When using unicellular green algae, for example, water is used as a medium or the medium shown in Example 22 is usually used for 25 hours.
The cells may be cultured and grown at 33°C for 24 hours to 48 hours, preferably 30 hours to 40 hours, and then collected.

Next, the culture medium or medium used for culturing and propagation is removed from the collected bacterial bodies by washing and rinsing several times with water etc.
Furthermore, the bacterial cells are dispersed in an aqueous solution containing a polymerized selenium substrate and/or a water-soluble selenium compound, and selenium is accumulated in the bacterial cells as polymerized selenium.
When this reaction is induced, the bacterial cell concentration is adjusted to be higher than the biological space to completely stop the consumption of the polymerized selenium substrate by bacterial cell growth, and the consumption of the polymerized selenium substrate is exclusively controlled. It is necessary to direct it to molecularization reactions. Preparing the bacterial cell concentration to be higher than this biological space means that even if the bacterial cell suspension (reaction liquid) essentially contains components that allow the growth of the facet, the bacteria in a certain volume of the suspension are This means that the bacteria are prepared under conditions in which the cells are substantially unable to proliferate any further, that is, the concentration is higher than the steady-state concentration of the cells in the cell growth curve. for example,
109 cells/ml or more for yeast, 108 cells/ml for lactic acid bacteria
m i, , for chlorella it is 109 q/m or more, so for yeast it is 1 to 5 x 10 q/m during the reaction.
m 12 , 1 to 5 x 108 bacteria/m for lactic acid bacteria! For Chlorella, it is sufficient to use a bacterial cell concentration of 1 to 5 x 10'/mE, and in practice, the bacterial cell concentration in a suspension of these microorganisms is measured by the OD value using a spectrophotometer.
The measurement error of the OD value may be ±10% when the bacterial cell concentration is in the stationary phase of the growth curve of the target bacterial cells. Furthermore, in the case of mold, it is impossible to measure by OD, so the bacterial cells may be suspended in a reaction solution smaller than the amount of the culture solution.

Next, the bacterial cells are dispersed in an aqueous solution containing an organic compound and/or a water-soluble selenium compound that can serve as a substrate for polymerizing selenium, thereby accumulating polymerized selenium within the bacterial cells.

Water that contains organic compounds and water-soluble selenium compounds that can serve as selenium polymerization substrates in the reaction solution? Solution 8 can be prepared by making an aqueous solution of both and dispersing the bacteria at a concentration higher than the above-mentioned biological space at the same time, or by sequentially dispersing the bacteria into an organic compound aqueous solution. It does not matter whether the bacterial cell concentration is higher than the biological space and then dispersed in an aqueous solution of a water-soluble selenium compound, or by reversing the order of dispersion. Any process that involves dispersion and contact with a liquid may be used.

First, organic compounds that can serve as selenium polymerization substrates are essential components for operating high-energy phosphoric acid compound synthesis mechanisms, such as glycolytic reaction mechanisms and TCA cycle reaction mechanisms, which are intracellular reaction mechanisms. That is, any organic compound that can serve as an energy substrate for bacterial cells may include sugars and/or amino acids. Examples of the sugar include glucose, fructose, galactose, sucrose, maltose, and trehalose, with glucose being particularly preferred. The amino acid may be a glucogenic amino acid such as leucine, isoleucine, or serine, and leucine or isoleucine is particularly preferred. The organic compound aqueous solution serving as the reaction solution preferably contains at least the above-mentioned sugar and/or amino acid and, if necessary, a phosphate or the like as a buffer.

Furthermore, if adenine or adenosine is added to the above system as a source of high-energy phosphoric acid compounds if necessary, polymerized selenium is more effectively accumulated in the bacterial cells.

When using the organic compound aqueous solution in the reaction described above, the pH of the aqueous solution is preferably adjusted to pH 4 to 7, particularly pH 5 to 6. When a buffer such as a phosphate salt is used, the buffer may be adjusted to a preferred pH.

Regarding the quantitative relationship of each component of the organic compound aqueous solution,
Sugar and amino acid may be used alone or in combination with respect to the total amount of the organic compound aqueous solution, and sugar is preferably contained in an amount of 0.3% to 2%, and amino acid is contained in an amount of 0.3% to 2%, and as necessary. The phosphate buffer is 0. OIM to 0.3M may be used, and when adenine and adenosine are added, 0.001% to 0.02% may be used.

Next, the water-soluble selenium compound, which is a selenium source that can be used in the present invention, is a suitable water-soluble selenium compound that can permeate the cell membrane of the target bacterial cell and be converted to polymerized selenium by binding to proteins within the bacterial cell. Any compound may be used, such as inorganic selenium such as selenite (H, SeO), selenium dioxide (SeOz), and selenic acid (HzSeO4).
In particular, it is preferable to use selenite. The amount of the water-soluble selenium compound used in the reaction may be adjusted so that the selenium concentration in the reaction solution is 2 PPm or more, preferably 10 to 1100 PP.

As mentioned above, the bacterial cells are simultaneously or sequentially dispersed in the reaction solution and reacted by shaking. It is preferable to react for 4 hours or more, especially 8 to 20 hours.

After the reaction, microbial cells containing polymerized selenium are obtained by collecting the bacteria by centrifugation or the like, and washing the collected cells several times with water. Further, the obtained polymerized selenium-containing bacterial cells may be stored by freeze-drying or the like, if necessary.

The present invention provides a method for obtaining efficient and stable polymerized selenium-containing microbial cells as described above.

<Examples> The present invention will be specifically described below with reference to Examples, but the present invention is not limited by these in any way.

Example 1 fII mother culture and creation of growth curve 100 ml of YPG medium adjusted to pH 5 (glucose 4%, peptone 1%, yeast extract 0.5%, potassium phosphate 0.5%, magnesium sulfate 0.2%) After dispensing it into a 500 ml Erlenmeyer flask and sterilizing it at 121°C for 15 minutes,
One platinum loop of P-2326) was planted and cultured with shaking at 30°C for 24 hours to use as a seed. After that, molasses medium adjusted to pH 5 (molasses 10% (contains 4% as sucrose), urea 0.5
%, 75% phosphoric acid 0.1%, zinc sulfate 0.0003%)
100rr+j! 5 ml of inoculum was inoculated and cultured at 30°C for 30 hours to determine the growth rate, and the growth curve is shown in Figure 1.

As described above, since the medium contained substantially no selenium, bacterial cell growth was extremely good.

Example 2 Effect of culture time and reaction (shaking) time on selenium uptake Satucharomyces cerevisiae F under the same conditions as Example 1
TY-3 cells were cultured for 8,1216 20 24
．． Centrifugation was performed every 28 hours (300 rpm,
After collecting the bacteria (for 15 minutes) and washing three times with 100 mf of water, the bacterial cell concentration was adjusted to 1 to 1.6 x 109 cells/ml to achieve a bacterial cell concentration higher than the biological space.
The bacterial cells were dissolved in 0.1M phosphate buffer containing 1% glucose (
pH6) 50mf! 5 mg of selenite (selenium concentration: 60 ppm) was added, and the mixture was shaken at 30°C to react.

Shaking (reaction) time: 2, 4, 16.24 hours The bacterial cell concentration was measured at each mouth, and the bacteria were collected by centrifugation.
After washing twice with 00 mf water, the cells were freeze-dried, and the concentration of selenium incorporated into the bacterial cells was measured.

The results are shown in Table 1.

In addition, to measure the bacterial cell concentration, dilute the reaction solution appropriately with water and add 660
Absorbance was measured at nm, and the concentration of selenium incorporated into the bacterial cells was measured by atomic absorption spectrometry after weighing approximately 100 mg of dry bacterial cells and wet ashing. In Examples, the bacterial cell concentration and the selenium concentration taken into the bacterial cells were all measured using the same method.

5mf of Saccharomyces cerevisiae FTY-3 inoculum cultured in the YPG medium of Example 1 with 5mg of selenite (30ppm as selenium concentration) added in advance to 100mj2 of influence molasses medium! , and cultured at 30"C for 30 hours.
Measure the bacterial cell concentration every 16, 20, 24, and 30 hours,
Collect bacteria by centrifugation, and then add 100ml of water to the cells.
After washing twice and freeze-drying, the concentration of selenium incorporated into the bacterial cells was measured. The results are shown in Table 2. The amount of dry bacterial cells after 30 hours of culture was 690 mg.

From the above, although almost no bacterial cell proliferation was observed over the course of the reaction time, the selenium uptake concentration of the bacterial cells at each culture time increased with the reaction time, and when using Satucharomyces cerevisiae, the time period of culture was approximately 8 to 20 hours. It was most suitable to carry out the reaction using a nutrient cell.

Reference example: From the above, when cultured in a medium to which selenite has been added in advance, selenium uptake is improved. It was found that the selenium uptake rate of bacterial cells was extremely low.

Example 3 Effect on selenium uptake when using selenium dioxide Saccharomyces cerevisiae F.
TY-3 was cultured at 30°C for 16 hours, and cultured at 1100 m
After washing the bacterial cells obtained in step f three times with 100 ml of water, the bacterial cell concentration was adjusted to 1.5 x 109 cells/m2 to achieve a bacterial cell concentration higher than the biological space, and the bacterial cells were reduced to 1%. 0.1M phosphate buffer (pH 6) containing glucose
50mj! 4. Selenium dioxide suspended in water and dissolved in water.
Add 15mg (50ppm as selenium concentration) and
The reaction was allowed to occur by shaking at 0°C for 20 hours, and the bacteria were collected by centrifugation. The cells were washed twice with water and then freeze-dried to obtain 1.19 g of dried bacterial cells (selenium concentration in the bacterial cells: 142QPPm) (uptake rate 51.2%).

Example 4 Effect on selenium uptake when using selenate
TY-3 was cultured at 30°C for 16 hours, and culture i! 100
100mj of bacterial cells obtained from mj2! After washing three times with water, the bacterial cell concentration was reduced to 1.5 x 10'/m! The cells were prepared in a 0.1 M phosphate buffer 1 containing 1% glucose (
pH 5) 3.7 mg of selenic acid (40 ppm as selenium concentration) dissolved in water was suspended in 50 mf.
The reaction was allowed to occur by shaking at 0°C for 20 hours, the bacteria were collected by centrifugation, the cells were washed twice with 100 ml of water, and then freeze-dried.
440 ppm) (uptake rate 80%).

Example 5 Effect of no addition of phosphoric acid on selenium uptake Satucharomyces cerevisiae F under the same conditions as Example 1
TY-3 was cultured at 30°C for 16 hours, and 100 m
j! 100mj of the bacterial cells obtained! After washing three times with water, the bacterial cell concentration was adjusted to 1.5 x 10' cells/ml to achieve a bacterial cell concentration higher than the biological space, and the bacterial cell concentration was adjusted to 0.5%, 1%, and 2%. glucose at each concentration of 5
0 ml, add 5 mg of selenite (60 ppm as selenium concentration), react by shaking at 30°C for 18 hours, measure the bacterial cell concentration, collect the bacteria by centrifugation, and further collect the bacterial cells. After washing twice with 100 ml of water, the cells were freeze-dried, and the concentration of selenium incorporated into the bacterial cells was measured. The results are shown in Table 3 Table 3 Bacterial cell concentration (ODbho) and intrabacterial selenium concentration (ppm)
The bacterial cells obtained from 100ml of the nutrient solution were mixed with 100mf of water.
After washing twice, the bacterial cell concentration was adjusted to 1.6 x 10' cells/ml to achieve a bacterial cell concentration higher than the biological space, and the bacterial cells were added to a 0.1 M phosphate buffer (pH 5) containing 1% glucose. ) 50mf, 0.02% each of adenosine and adenine, and no additives were prepared. 5mg of selenite (selenium concentration: 60ppm) was added to each, and the mixture was shaken at 30°C for 20 hours to react. , measure the bacterial cell concentration, collect the bacteria by centrifugation, and further collect 100 bacterial cells.
m/! After washing twice with water, the cells were freeze-dried, and the concentration of selenium incorporated into the cells was measured. The results are shown in Table 4.

From the above, selenium was successfully incorporated into the bacterial cells even without the addition of phosphoric acid.

Example 6 Effect of addition of adenine and adenosine on selenium uptake Satucharomyces cerevisiae F under the same conditions as Example 1
TY-3 was cultured at 30°C for 20 hours, and as described above,
The addition of adenine and adenosine made the uptake of reselene into the bacterial cells more effective.

Example 7 Effect of selenium concentration on selenium uptake The same conditions as Example 1, Calomyces cerevisiae F
TY-3 was cultured at 30°C for 20 hours, and 100 m
jl! After washing the obtained bacterial cells three times with 100 mf of water, the bacterial cell concentration was adjusted to 1.7 x 10 q/ml to reach the biological space concentration, and the bacterial cells were soaked in 1% glucose. 01M phosphate buffer (pH 5) containing 5
Suspend in 0 ml and add Og selenite (Og as selenium concentration).
ppm), 1mg (12.5pp as selenium concentration)
m), 2mg (25pp++ as selenium concentration
), 4 mg (50pp+i as selenium concentration
), 6mg (75ppm as selenium concentration)
, 8mg (100 ppm as selenium concentration)
The mixture was prepared by adding each of
After washing twice with water, the cells were freeze-dried, and the amount of dried bacterial cells and the concentration of selenium incorporated into the bacterial cells were measured. The result is the fifth
Shown in the table.

From Table 5 and above, the amount of selenite added to the reaction solution is 1 mg to 8
mg (By using a selenium concentration of 12 PPm to 10,100 ppm, selenium was well incorporated into the bacterial cells.

Example 8 Effect of PH of reaction solution on selenium uptake The same conditions as Example 1, Calomyces cerevisiae F
TY-3 was cultured in 6 flasks, and culture solution 1
00mj! After washing the obtained bacterial cells three times with 100 ml of water, the bacterial cell concentration was adjusted to 1.8XIO9 cells/ml to achieve a bacterial cell concentration higher than the biological space,
The bacterial cells were incubated at pH 3, 4, 5, 6, 7 containing 1% glucose.
, 8 0 and 1 M phosphate buffers prepared respectively in the range of 5
0ml, add 5mg of selenite (60ppm as selenium concentration), shake at 30°C for 20 hours to react, collect the bacteria by centrifugation, and further collect 100+n of bacterial cells.
After washing twice with water from Step 1, the cells were freeze-dried, and the concentration of selenium incorporated into the cells was measured. The results are shown in Table 6.

From Table 6 and above, selenium was successfully incorporated into the bacterial cells by adjusting the pH of the reaction solution to pH 4 to 6.

Example 9 Effect of reaction (shaking) temperature on selenium uptake Satucharomyces cerevisiae F under the same conditions as Example 1
TY-3 was cultured in 5 flasks, culture solution 1
After washing the bacterial cells obtained from 00rr+1 three times with 100 ml of water, the bacterial cell concentration was adjusted to 1.5 x 10' cells/ml to make the bacterial cell concentration higher than the biological space, and the bacterial cells were washed with 100 ml of water three times. Suspended in 50mj2 of 0.1M phosphate buffer (pH 5) containing % glucose, 5mg of selenite (
60 ppm) was added as a selenium concentration, and 20°C and 25°C were added.
, 30°C, 35°C, 20°C in each temperature range of 940°C.
Shake for a few hours to react, collect the bacteria by centrifugation, and further collect the bacteria at 100 mJ! After washing twice with water, freeze-drying,
The concentration of selenium taken into the bacterial cells was measured. The results are shown in Table 7.

From Table 7 and above, the reaction (shaking) temperature during reaction is 25
By keeping the temperature between 35°C and 35°C, selenium was well incorporated into the bacterial cells.

Example 10 Effect of adenine addition concentration on selenium uptake Satucharomyces cerevisiae F under the same conditions as Example 1
TY-3 was cultured in 6 flasks, and culture if
flroomj! After washing the obtained bacterial cells three times with 100 ml of water, the bacterial cell concentration was adjusted to 1.5 x 109 cells/m2.
j2 to bring the bacterial cell concentration above the biological space, suspend the bacterial cells in 50 mf of 0.1 M phosphate buffer (pH 5) containing 1% glucose, and add adenine to 0.
%, 0.001%, 0.003%0.01%, 0
．． 03% and 0.1%, added 6 mg of selenite (72 ppm as selenium concentration), reacted by shaking at 30°C for 20 hours, and collected bacteria by centrifugation. Furthermore, the bacterial cells were washed twice with 100 ml of water and then freeze-dried, and the concentration of selenium incorporated into the bacterial cells was measured. The results are shown in Table 8.

(Margin below) From Table 8 and above, when adding adenine to the reaction solution, selenium was well incorporated into the bacterial cells by adjusting the concentration to 0.003% to 0.1%. .

Example 11 Effect of phosphoric acid addition concentration on selenium uptake The same conditions as Example 1, Calomyces cerevisiae F
TY-3 was cultured in 6 flasks, and culture solution 1
After washing the bacterial cells obtained from 00mj2 three times with 100 ml of water, the bacterial cell concentration was adjusted to 1.6 x 10' cells/m1 to reach a bacterial cell concentration higher than the biological space, and the bacterial cells were OM 0.00 with 1% glucose
3M, 0. OLM 0.03M, 0.1M,
Phosphate buffer solution (pH 5) prepared to 0.3 M each
50 mjl! 5 mg of selenite (60 ppm as selenium concentration) was added to each, shaken at 30°C for 20 hours to react, collected by centrifugation, and washed twice with 100 mj2 of water. The cells were freeze-dried and the concentration of selenium incorporated into the cells was measured. The results are shown in Table 9.

From Table 9 and above, when a phosphate buffer was added to the reaction solution, selenium was well incorporated into the bacterial cells by adjusting the concentration of phosphoric acid to 0.003 M to 0.3 M.

Example 12 500 mf of each Satucharomyces cerevisiae FTY-3 inoculum cultured in a YPG medium for 24 hours in a flask under the same conditions as in Example 1 was adjusted to pH 5 using four 30 A jar fermenters and incubated at 120°C. Plant in 2ON molasses medium sterilized by heat for 30 minutes, and place at 32°C1 aeration 301/
The cells were cultured for 16 hours at a stirring speed of 300 rpm. Bacteria were collected using a continuous centrifuge (12,017 hours), and about 20 ml of water was added to suspend the bacteria, followed by centrifugation to wash the bacteria. After repeating this operation three times, the bacterial cell concentration was increased to 2.5X1.
The microbial cell concentration was adjusted to 0.09 cells/mj2 to achieve a bacterial cell concentration above the biological space, suspended in 5 f of 0.03M phosphate buffer (pH 5) containing 0.5% glucose, and 0.4 g of selenite ( Addition of selenium concentration (48 ppm) and ventilation rate 10! /min, a stirring speed of 200 rpm, and a shaking (reaction) temperature of 30''C. After 18 hours, the bacteria were collected by centrifugation, and further washed three times with 2ON water to obtain 3.5 kg of wet bacteria. After that, 7! of water was added, stirred and dispersed, and heated at 90°C for 15 minutes. Using a Kochiwa spray dryer, air was blown at 140°C, exhaust air was 60°C, and liquid was sent at 442°C/hour. Spray dry and dry powder1.
1 kg was obtained (moisture 4.1%, selenium concentration inside the bacteria 191
0ppm).

Reference Example 5 g of bacterial cells obtained in Example 12 were suspended in 50 mf of water, adjusted to pH 7 with IN caustic soda, crushed with a positron crusher, further heated at 95°C for 10 minutes, Centrifuge at 10,000 rp+n for 15 minutes and remove supernatant 3.
8 ml was obtained (selenium concentration 174 PPm). Kamisume 10m
After freeze-drying j2, it was dissolved in 2 mf of water, of which 1
The elution pattern and selenium distribution using a 100 m42 column (2X32cs+) of Sephadex G50 were investigated using mf. As a result, as shown in Figure 2, more than 85% of selenium was present in the polymer fraction.

Example 13 Effect of glucose concentration and reaction (shaking) time on selenium uptake Satucharomyces cerevisiae F under the same conditions as Example 1
Culture solution 600 obtained by culturing TY3 at 30'C for 16 hours
m/! After centrifuging to collect bacteria (300 Orp for 15 minutes) and washing three times with 500 mj2 water, the bacterial cell concentration was adjusted to 1.4 x 10' cells/ml and the bacterial cell concentration in the biological space was determined. and suspended in 0.1M phosphate buffer (pH 5) and amplified to 300mj2, of which 50mj! 500mf! Dispense 4.1 mg of selenite (selenium concentration: 50 ppm) into a Erlenmeyer flask.
) and glucose O%, 0.1% 0.3%, 1% 12%,
3% and 5% were added, and the mixture was shaken and reacted at 30°C. After that, the bacterial cell concentration was measured at each 4,1624th hour, and the bacteria were collected by centrifugation.
After washing twice with water, the cells were freeze-dried, and the concentration of selenium incorporated into the dried cells was measured. The results are shown in Table 10.

From the above, when glucose was used as the organic compound that is the polymerization substrate for selenium, the concentration of addition was preferably 0.3% or more.

Example 14 Effect of tR#I type on selenium uptake Satucharomyces cerevisiae F under the same conditions as Example 1
TY-3 was cultured at 30"C for 16 hours, and 100 m
After washing the bacterial cells obtained from ff1 three times with 100 mf of water, the bacterial cell concentration was adjusted to 1.8 x 10" cells/m2 to achieve the bacterial cell concentration in the biological space, and the bacterial cells were reduced to 1%. Contains glucose, fructose, galactose, shakerose, maltose, and trehalose, respectively.
Suspended in 50 mffi of 1M phosphate buffer (pH 5), 5 mg of each selenite (60 ppm as selenium concentration)
! In addition, the reaction was allowed to occur by shaking at 30°C for 20 hours, the bacteria were collected by centrifugation, and the cells were washed twice with 100 ml of water and freeze-dried to determine the amount of dried cells and the amount taken into the cells. The selenium concentration was measured in each case. The results are shown in Table 1I.

(Margins below) From Table 11 and above, we can see that in addition to glucose, fructose, sucrose, maltose, and trehalose can be used as organic compounds that are polymerization substrates for selenium to better incorporate selenium into bacterial cells. I was able to do that.

Example 15 Effect of amino acid addition on selenium uptake Satucharomyces cerevisiae F under the same conditions as Example 1
TY-3 was cultured at 30°C for 16 hours, and 100 m
j! After washing the bacterial cells obtained from the above three times with 100 ml of water, the bacterial cell concentration was adjusted to 1.8 XIO'' cells/m2 to reach a bacterial cell concentration higher than the biological space, and the bacterial cells were washed with 0.0 ml of water. Suspended in 50 ml of 0.1 M phosphate buffer (pH 5) containing 5% each of leucine, isoleucine, serine, glutamic acid, and asparagine, and 5% each of selenite,
g (60 ppm as selenium concentration) was added and reacted by shaking at 30°C for 20 hours, and the bacteria were collected by centrifugation.The cells were further washed twice with 100 mff of water, and then freeze-dried. The amount of selenium taken into the cells and the concentration of selenium taken into the cells were measured. The results are shown in Table 12.

From Table 12 and above, by using leucine and isoleucine as organic compounds that are polymerization substrates for selenium, it was possible to successfully incorporate selenium into the bacterial cells.

Example 16 When the microorganism is Candida krusei
i) YP 156-”25A (IF○-0841)
Cultivate in G medium at 30°C for 20 hours, and add 100 mj of culture solution.
! After washing the bacterial cells obtained from the above three times with 100rr+1 water, the bacterial cell concentration was adjusted to 1.2 x 10' cells/ml to obtain the bacterial cell concentration in the biological space, and the bacterial cells were reduced to 1%. 0.1M phosphate buffer containing glucose (pH
6) Suspend in 25mA, add 1.3mg of selenite (30ppm as selenium concentration), shake at 30"C for 20 hours to react, collect the bacteria by centrifugation, and add the bacteria to 100ml of water. After washing twice with water, the cells were freeze-dried, and the amount of dried bacterial cells and the concentration of selenium incorporated into the bacterial cells were measured.The results are shown in Table 13.

From Table 13 and above, selenium was successfully incorporated into the bacterial cells even when yeast of the genus Candida was used.

Example 17 When the microorganism is Torulaspora delbrellakii
radelbrueckii) 154-CA (I
FO-1129) was cultured in YPG medium at 30°C for 20 hours, and the bacterial cells obtained from 100mf of the culture solution were cultured in 100mf.
After washing 3 times with water of f, the bacterial cell concentration was adjusted to 1.8 x 10" cells/m1 to make it the bacterial cell concentration of biological space 1, and the bacterial cells were mixed with 0.1 microorganisms containing 1% glucose.
M phosphate buffer (pH 6) 25m/! Add 1.3 mg of selenite (selenium concentration: 30 ppm), shake at 30°C for 20 hours to react, collect the bacteria by centrifugation, and add the cells twice with 100 ml of water. After washing and freeze-drying, the amount of dried bacterial cells and the concentration of selenium incorporated into the bacterial cells were measured. The results are shown in Table 14.

From Table 14 and above, selenium can be incorporated into the bacterial cells well even when Torulaspora yeast is used.Example 18 When the microorganism is Lactobacillus casei
Leuconostoc medium (2% glucose 0.5% yeast extract, 0.5% peptone: 12
Sterilize at 0°C for 15 minutes) and culture at 30°C for 20 hours. After washing three times with water, the bacterial cell concentration was adjusted to 1.2 x 10"/m! to achieve a bacterial cell concentration higher than the biological space, and the bacterial cells were washed with 0.1 M water containing 1% glucose. Suspended in phosphate buffer (pH 6) 25 mA, selenite 1.3
mg (30 ppm as selenium concentration) and heated at 30°
The cells were shaken at C for 20 hours to react, collected by centrifugation, and the cells were washed twice with 100 ml of water, then freeze-dried, and the amount of dry cells and the concentration of selenium incorporated into the cells were measured. did.

The results are shown in Table 15.

(Margin below) Table 15 From Table 15 and above, selenium could be successfully incorporated into the bacterial cells even when lactic acid bacteria were used as the microorganism.

Example 19 When the microorganism is Lactobacillus andophilus
lIus acidphilus) B-105]
(l FO-3953) was cultured at 30°C for 20 hours in Leuconostoc medium adjusted to pH 7, and the culture solution 10
After washing the bacterial cells obtained from 0mf three times with 100 ml of water, the bacterial cell concentration was adjusted to 1.1XIO'' co/mn to achieve a bacterial cell sensitivity higher than the biological space, and the bacterial cells were 1.3 mg of selenite was suspended in 25 mj! of 0.1 M phosphate buffer (pH 6) containing 1% glucose.
(Selenium concentration: 30 ppm), stirred at 30°C for 20 hours to react, collected bacteria by centrifugation, and then heated the facepiece to 100 mJ! After washing twice with water, the cells were freeze-dried, and the amount of dried bacterial cells and the concentration of selenium incorporated into the bacterial cells were measured. The results are shown in Table 16.

Table 16 Example 20 When Aspergillus niger is used as a microorganism, Aspergillus niger
YM medium (1% glucose, 05% peptone,
0.3% yeast extract, 0.3% malt extract: 12
Sterilize at 0°C for 15 minutes) and culture at 25°C for 30 hours.
After washing twice, the bacterial cells were washed with 0.1M solution containing 1% glucose.
Suspend in 15mff of phosphate buffer (pH 6), add 1.25mg of selenite (50ppm as selenium concentration), shake at 30°C for 18 hours to react, collect by centrifugation, and further The cells were washed twice with 100 ml of water and then freeze-dried to obtain 0.48 g of dried cells. The selenium concentration taken into the bacterial body was 920 ppm (uptake rate 46%).
)Met.

Example 21 When the microorganism is Aspergillus awamori
se awallori) N04A (IFO-40
33) was cultured in YM medium adjusted to pH 7 at 25°C for 30 hours, and the bacterial cells obtained from 100 ml of culture solution were
mj! After washing three times with water, the cells were washed with 0.1M phosphate buffer 1 (pH 6) containing 1% glucose at 25mj! 2.5 mg of selenite (60P as selenium concentration)
pm), reacted by shaking at 30'C for 18 hours, collected the bacteria by centrifugation, washed the cells twice with 100ml of water, and freeze-dried to obtain 0.62g of dried cells. I got it. The selenium concentration taken into the bacterial body is 2230 ppm.
(uptake rate 70%).

From the above, even when mold was used as the microorganism, it was possible to successfully incorporate selenium into the bacterial cells.

Example 22 When the microorganism is Chlorella vulgaris
allis) E-25 in 2% glucose, 0.4% urea, 0.1% phosphate, 0.1% magnesium sulfate, 0.
Cultured at 30°C for 30 hours in a medium containing 0.3% ferrous sulfate and 0.1% calcium chloride, and the culture solution was 100mj! 100m/! of bacterial cells obtained from After washing three times with water, the bacterial cell concentration was adjusted to 2 x 10' cells/ml to achieve a bacterial cell concentration higher than the biological space, and the bacterial cell concentration was adjusted to 0.
0.1M phosphate buffer (pH 6) containing 5% glucose
25 mf, add 5 mg of selenite (120 ppm as selenium concentration), shake at 30°C for 18 hours to react, measure the bacterial cell concentration, collect the bacteria by centrifugation, and further collect the bacterial cells. After washing twice with 100 mj2 of water, the cells were freeze-dried, and the concentration of selenium incorporated into the cells was measured. The results are shown in Table 17.

(Margins below) Table 17 As shown above, even when unicellular green algae was used as the microorganism, selenium could be successfully incorporated into the bacterial cells.

<Effects of the Invention> The purpose of the production method of the present invention is to culture and proliferate bacterial cells in a substantially selenium-free medium without adding selenium in advance, so that there is no growth inhibition due to selenium during culturing, which is different from the conventional technology. To provide a method in which the growth rate of bacterial cells is faster than in the case of culturing and growing in a medium to which selenium has been added in advance, and the growth rate is also excellent.

In addition, in the present invention, the bacterial cells are dispersed in the reaction solution after sufficiently culturing and propagating Kokutai in a medium, and after collection, the bacterial cells can be reduced to four, so the reaction system can be made smaller. Therefore, the amount of waste liquid after the reaction can be reduced, and since the reaction liquid does not contain metals other than selenium, a method is provided that simplifies waste liquid treatment.

Furthermore, the present invention allows the bacterial cells to propagate appropriately, and after collecting the bacteria, washes with water to remove culture medium components, and then adjusts the bacterial cell concentration to II, preferably above the biological space, to stop the culture and proliferation.
Since the bacterial cells are then dispersed in the reaction solution, there is naturally no need for bacterial cell proliferation during the reaction, and it is possible to increase the concentration of selenium, which inhibits bacterial cell growth, in the reaction solution. Moreover, the concentration of polymerized selenium that can be taken up and accumulated in the bacterial cells is high, and the present invention provides the production of bacterial cells containing a high concentration of polymerized selenium.

[Brief explanation of drawings]

Figure 1 shows the growth curve of bacterial cells determined in Example 1, and Figure 1 shows the growth curve of bacterial cells determined in Example 1.
The figure shows the elution pattern and selenium distribution determined in Example 12 Reference Example.

Claims (1)

[Claims] The bacterial cells obtained by culturing a microorganism capable of accumulating medium-polymerized selenium in the bacterial cells are brought to a bacterial cell concentration higher than the biological space, and the bacterial cells are treated with a polymerized selenium substrate. 1. A method for producing polymerized selenium-containing bacterial cells, which comprises accumulating polymerized selenium in the bacterial cells by dispersing them in an aqueous solution containing a certain organic compound and a water-soluble selenium compound. (2) The production method according to claim 1, wherein the microorganism capable of accumulating polymerized selenium within its bacterial cells is edible yeast, bacteria, mold, or unicellular green algae. (3) The edible yeast is Saccharomyces
omyces), Torulaspora (Torulasup)
ora), Torulopsis, Mycotorula, Candida, Hansenu
The method according to claim 2, wherein the yeast belongs to the genus la). (4) The production method according to claim 1, wherein the water-soluble selenium compound is selenite, selenium dioxide, or selenic acid. (5) The production method according to claim 1, wherein the organic compound that is the polymerization substrate for selenium is a sugar and/or an amino acid. (6) Sugar is glucose, fructose, sucrose,
6. The method according to claim 5, wherein the amino acid is maltose or trehalose and the amino acid is leucine or isoleucine. (7) The method according to claim 5, wherein the organic compound aqueous solution that is the polymerization substrate for selenium contains at least sugar and/or amino acid and phosphate.