KR100434949B1 - Preparation method of yeast containing of organic Se-I complex - Google Patents

Abstract

PURPOSE: A preparation method of yeast containing organic selenium(Se)-iodine(I) complex is provided, which organic selenium(Se)-iodine(I) complex can prevent various diseases, and is useful in feeds, health food and medicines because it has no toxicity and high absorption rate. CONSTITUTION: The preparation method of yeast containing organic selenium(Se)-iodine(I) complex comprises the steps of: (1) seed-culturing Saccharomyces cerevisiae or Candida albicans; (2) inoculating the seed-cultured Saccharomyces cerevisiae or Candida albicans into GY medium containing 0.1 to 1.0 g/l of iodine and culturing the microorganism; (3) after the certain time of cultivation is passed, adding an optimal fed-batch medium containing selenium is added into the culturing medium, and culturing the microorganism; and (4) concentrating, destroying and drying the cultured Saccharomyces cerevisiae or Candida albicans containing the organic selenium(Se)-iodine(I) complex, wherein the Saccharomyces cerevisiae is Saccharomyces cerevisiae Ya(KCTC 7107), Saccharomyces cerevisiae Yb(KCTC 7919), Saccharomyces cerevisiae Yc(KCTC 7928), Saccharomyces cerevisiae Yd(KCTC 7965) or Saccharomyces cerevisiae Ye(KCTC 7963).

Description

Preparation method of yeast containing organic se-I complex}

The present invention relates to a method for producing an organic selenium (se) -iodine (I) complex using yeast.

As agriculture is intensively developed, the quality of trace minerals available in the soil is decreasing year after year, and thus, the trace minerals are insufficient in the crops produced in the soil. Therefore, in general, in order to compensate for the deficiency of trace minerals in feed grains and grassland, it is necessary to add inorganic trace minerals in the feed formulation.

Animal content of trace minerals is relatively low compared to other nutrients, but its importance is increasing due to its effects on animals and humans. Since trace amounts of trace minerals are often lacking in feedstocks of animals, it is important to consider the bioavailability of mineral sources in order to meet feed requirements. Recently, as a feed additive, bioavailability is higher than that of inorganic minerals, and due to the high digestive absorption rate, the amount of the animal can be supplied in a small amount, and since the body is absorbed, there is an increasing interest in environmentally friendly organic minerals.

Organic minerals are absorbed into the body through active absorption, which is used when amino acids or peptides are absorbed, rather than active transport, which is a common mineral absorption pathway when absorbed in the small intestine, thus preventing contention between minerals. Transported to promote intestinal absorption (Kim, YY and DC Mahan, J. Animal. Sci. , 14 (2) , pp 243-249, 2001). Kelly, MP and RF Power, J. Dairy Sci. , 78 (suppl . 1) , p237, 1995, contain 67% selenium in the cell cytosol in the form of a selplex. It is reported that 11% is present in mitochondria.

Selenium was discovered by Swedish chemist JJ Berelius in 1818 as the selenium found in the residues of sulfuric acid produced during the smelting process of metals. However, in 1957, Swartz and Foltz (Schwarz and Foltz), selenium is known as a substance that can prevent liver damage in rats, and later glutathione peroxidase (GSH-Px), which plays an oxidative role in the cytoplasm. As it was later discovered that it was a component of (Rotruck, JT, et al., Science , 179 , p588, 1973), selenium began to be recognized as an essential trace mineral. In particular, when selenium deficiency in the human body decreases the activity of GSH-Px, it does not effectively remove H ₂ O ₂ , an oxide produced during metabolic processes, causing cell dysfunction or cell destruction (Sies, H., C). , et al, FFES Lett. , 27 , pp 171-175, 1972; Eklow, L., et al., FESS Lett. , 127 , pp125-128, 1981). In addition, the deficiency of selenium in animals and humans causes various nutritional diseases. Major physiological changes include growth retardation, skin disease, hair loss, blindness, reproduction, and liver or muscle necrosis. White muscle disease in sheep and cattle is caused by necrosis of muscle tissue, and myoglobin in muscles is destroyed by oxides such as hydrogen peroxide (H ₂ O ₂ ) formed in the body due to deficiency of selenium. It is a good way to easily determine whether slaughtered slaughtered animals are slaughtered due to their lack of muscle redness and thinning of the muscle as a whole (Schwarz, K., et al., Med. Clin). North Amercia , 60 , p745-457, 1976). Inorganic selenium (selenite or selenate) is absorbed by simple diffusion, so its digestibility is generally 80% higher than organic selenium transported by active transport (Kim, YY and DC Mahan, J. Animal. Sci. , 14 (2) ) , pp 243-249, 2001). However, since most of the absorbed inorganic selenium is excreted again through the urine, the amount of selenium actually accumulated in the body is higher when fed organic selenium, which is less excreted in vitro (Kim, YY and DC). Mahan, J. Animal.Sci . , 14 (2) , pp 243-249, 2001).

Recently, a method of producing organic selenium by selenium-enriched yeast has become common. Sulfur-containing amino acids that constitute the body protein of yeast are collected when the yeast cell protein is collected after cultivating the yeast with a medium of selenium having chemical properties similar to sulfur. Since sulfur, a constituent of these compounds, is substituted with selenium, organic selenium containing selenium can be obtained instead of sulfate (Mahan, DC, Biotechnology in the feed industry , Proceedings of Alltech's 11th Annual symposium , pp257-267, 1995).

Kelly, MP and RF Power, J. Dairy Sci. , 78 (suppl . 1) , p237, 1995) found that 94% of selenium in selenium-enriched yeast is a combination of several forms of amino acids. Selenomethionine is the most representative substance.

Accordingly, many researches are being made to make organic selenium using yeast. These studies are focused on maximizing the amount of yeast produced and the amount of selenium introduced into the cells of the yeast.

Iodine was discovered by B. Kurta of France in 1811, and was named by JL Gayrusac the Greek iodes, meaning that the steam is purple. Iodine is used to synthesize thyroxine and tri-iodo-thyroxine, which, when deficient, leads to hyperthyroidism. Goiter is caused by a lack of iodine or an increase in thyroxine demand because the thyroid acts unreasonably to provide all of the required amount (Maeng, DC and YY Kim., J. anim. Sci. , 74 , pp2711). -2718, 1996)

Therefore, the present inventors first evaluate the resistance and adaptability to toxicity by the growth rate for each yeast strain according to the concentration of inorganic selenium and iodine in order to increase the accumulation rate of selenium and iodine in yeast cells, and as a result Based on the results of the evaluation of the characteristics of the selenium-iodine-producing strains selected, based on the results to determine the optimum conditions for the increase in organic conversion, the success in producing organic selenium-iodine complex using yeast Thus, the present invention has been completed.

Accordingly, it is an object of the present invention to develop a method for producing organic selenium-iodine complex, which is an environmentally friendly complex organic mineral, using high yield.

1 is a diagram showing the growth of cells in each culture using YM medium and GY medium in 5 L fermentation tank culture,

Figure 2 is a diagram showing the change in cell mass of Saccharomyces cerevisiae in the batch culture according to the glucose concentration change in a 5 L fermentation tank,

Figures 3a to 3c relates to the time-phase change of Saccharomyces cerevisiae by exponentially fed-batch culture in 5 L fermentation tank,

Figure 3a is a diagram when the specific growth rate of 0.15,

Figure 3b is a diagram when the specific growth rate is 0.2,

3c is a diagram of the specific growth rate, when 0.25,

4 is a diagram showing the results of the intermittent oily culture of adding glucose when the carbon source is depleted during the cultivation,

5 is a view showing the culture by adding iodine and selenium in a 5 L seedling fermentation tank,

6 is a diagram of 500 l intermittent oil price culture,

7 is a schematic diagram of a method for producing a liquid fermentation product as a dry powder.

In order to achieve the above object, the present invention provides a method for producing an organic selenium-iodine complex using yeast.

More specifically, the present invention relates to (1) Saccharomyces cerevisiae ( Saccharomyces cerevisiae ) or Candida albicans (candida albicans ) in the first step of culturing the seed, (2) Saccharomyces cerevisiae ( Saccharomyces cerevisiae) Or to incubate the seed of Candia albicans seedlings, two steps of inoculation and cultivation in iodine-added medium, and (3) after a certain period of time, an optimum milk medium containing selenium is added and cultured. Step 3, (4) Concentration, cell crushing and drying of Saccharomyces cerevisiae or Candida albicans cells containing selenium and iodine complexes as complex organic minerals. It provides a method for producing an organic selenium-iodine complex comprising the Saccharomyces cerevisiae ( Saccharomyces cerevisiae ) is Saccharomyces cerevisiae ( Sac charomyces cerevisiae ) Ya (KCTC7107, sold at the Korea Biotechnology Research Institute Genetic Center), Saccharomyces cerevisiae ) Yb (KCTC7919, sold at the Korea Biotechnology Research Institute Genetic Center), Saccharomyces cerevisiae ( Saccharomyces cerevisiae ) Yc (KCTC7928, sold at the Korea Biotechnology Research Institute Genetic Center), Saccharomyces cerevisiae ) Yd (KCTC7965, sold at the Korea Biotechnology Research Institute Genetic Center) or Saccharomyces cerevisiae ( Saccharomyces cerevisiae ) Ye (KCTC7963, sold at the Korea Biotechnology Research Institute Gene Center), more preferably Saccharomyces cerevisiae (YC) (KCTC7107, sold at the Korea Biotechnology Research Institute Gene Center) strains can be used. .

Yeast culture medium that can be used in the present invention includes all yeast culture medium, preferably using YM medium or GY medium, more preferably GY medium. In the present invention, the pilot in the yeast culture The scale may use 5 L, 500 L, or 5000 L fermenter, preferably 5 L fermenter. In addition, the ratio of glucose and yeast extract in the medium in the yeast culture is 40 to 80 per liter of culture medium: 1, preferably in a ratio of 60 to 70: 1.

On the other hand, glucose may be added to 5 to 20 g per 1 liter of culture, yeast extract may be carried out by adding 0.01 to 1.0 g per 1 liter of culture.

Hereinafter, the present invention will be described in detail.

Yugitae selenium using the yeast of the present invention method of producing an iodine complex of (1) to the MY process in one step of culturing the microorganisms in a celebrity busy (Saccharomyces cerevisiae) or Candida albicans (Candia albicans), (2) saccharide saccharide To incubate seed of Saccharomyces cerevisiae or Candia albicans , two steps of inoculation and incubation in iodine-added medium, (3) Three steps of cultivation and addition of optimum oil medium (4) Concentration of Saccharomyces cerevisiae or Candida albicans cells containing selenium and iodine complex as complex organic minerals And four steps of drying to powder.

Specifically, in the first step, in my process serenity busy in the Saccharomyces (Saccharomyces cerevisiae) or Candida albicans in order to culture a seed of (Candia albicans) into the YMP medium in a test tube and inoculated with a colony of the agar plates in a shaking incubator Incubate for 12 to 48 hours at 25 to 35 ° C. and 120 to 170 rpm. Inoculate 1-20% (v / v) of this culture in a Erlenmeyer flask containing YMP medium for 6 to 24 hours under the same conditions as above. Incubate and use as inoculation bacteria.

In the second step, saccharose as MY access celebrity busy in (Saccharomyces cerevisiae) or Candida albicans (Candia albicans) microorganisms is the culture the initial yeast culture iodine in the range of 0.01 to 1.0 g / medium ℓ, the preferred to the culture of 0.4 to 0.6 g / l, more preferably 0.5 g / l (the feed concentration of iodine in the medium may be 50 to 5000 ppm, preferably not more than 2000 ppm), in the first step After inoculating 1 to 20% of the cultured inoculum, it is incubated for 6 to 48 hours under conditions of 25 to 35 ° C., aeration amount of 1 to 5 vvm, and agitation speed of 10 to 900 rpm at slightly acidic to neutral pH. At this time, the stirring speed is adjusted according to the size of the fermenter.

In the third step, the oil-containing medium containing selenium is added, and the medium may additionally contain glucose, yeast extract and sodium sulfate in addition to selenium as a constituent of the medium.

As the inorganic selenium among the selenium sources that can be used in the present invention, all compounds containing selenium and selenium may be used, preferably sodium selenite (Na ₂ SeO ₃ ).

The feed concentration of selenium in the oil medium may be 50 to 6000 ppm, preferably 500 ppm.

As a source of sulfate which can be used in the present invention, all compounds containing sulfates such as sodium sulfate, potassium sulfate, ammonium sulfate can be used, but preferably sodium sulfate is used. do.

Selenium-containing oil medium is added after 20 to 40 hours of cultivation, and further cultured for 20 to 30 hours after the addition of the oil-containing medium containing selenium. do.

In the fourth step, in order to concentrate, cell crush and dry the cells containing the complex organic minerals, the concentration is concentrated using a membrane separation filtration device, cell crushing using a cell crusher, and lyophilization using a lyophilizer. Yeast containing the material, ie selenium-iodine complex, is prepared.

In the present invention, in order to cultivate the yeast, a batch culture is used, and preferably a fed-batch culture is performed.

In addition, exponential fed-batch culture or intermittent fed-batch culture may be performed during the oil value culture, and preferably, intermittent oil value culture.

The present invention is described in more detail based on the following examples and experimental examples, but the present invention is not limited thereto.

Reference Example 1. MBRT (Methylene blue reduction time) measurement

The concentration of extracellular selenium was according to the method of Nagoda withana, et al. (Nagodaawithana, T. et al., U. S. Patent, No. 4,530, 846, 1985).

first, 7 ml of reducing solution (Reducing solution; % 1-thioglycerol / 0.20 N phosphate buffer, pH 5.5) was added and mixed for several seconds. After standing for 3 minutes, nitrogen gas was injected, and 100 µl of 2% methylene blue solution was added to control the disappearance of the blue color when mixing. Organic conversion was evaluated by comparing

Reference Example 2 Iodine Determination

Iodine quantification ATSM (Standard test methods for I and Br ion in brackish water, seawater, and brines. D3869, 1995).

First, Saccharomyces cerevisiae 1 kind The precipitate (culture medium 1 ml) was washed three times with 0.85% sodium chloride solution, and 5 ml of a mixed solution of 5% alcohol and 1% sodium chloride was added thereto, and dried in a hot air dryer at 60 ° C. for 24 hours. After drying, homogenate for 30 minutes with an ultrasonic homogenizer to completely crush the yeast strain, and then completely acidify by adding 1 ml of hydrochloric acid to the supernatant obtained by centrifuging the crushed yeast homogenate at 3000 rpm and 5 minutes. Solution (KNO₂1 ml), and again add carbon tetrachloride (CCl)₄1 ml of the solution was added to measure the optical density in an ELISA reader at 530 nm.

Reference Example 3. Selenium Determination

The total selenium concentration was measured by placing 1 g of the sample in a beaker, injecting 20 ml of nitric acid (HNO ₃ ) and 1 ml of phosphoric acid (H ₃ PO ₄ ), and leaving it overnight, then halving the sample liquid on a hot plate. After heating and evaporating to a decrease, the resultant was filtered through No. 6 filter paper, applied to a 100 ml volumetric flask, and measured using an ICP emission spectrometer (JIPN, Jobin Yvon, France).

Example 1 Yeast Strain Selection

To select strains to be used in this study, Saccharomyces cerevisiae, Candida albicans from the Korea Center for Biotechnology Research In addition to the strain, 18 yeast strains (yeast strain) were distributed and used for selection.

Frozen frozen strains were thawed and incubated for 24 hours in a flat medium, and the resulting colonies were returned to 10 ml of YM medium (YM media: 3 g of yeast extract, 3 g of malt extract, 5 g of peptone, and 20 g of glucose). After incubation for 24 hours, 50% glycerol (Glycerol) and one-to-one mixed and stored at -80 ℃ deep freezer (Deep freezer).

In this study, the results of the basic investigation (Rose, AH, FA, 1980) and the selection medium for each species were selected to verify the cation utilization efficiency of each species for the selection of high organic conversion rate of selenium and iodine. Considering the composition, cell weight, temperature, pH, etc. of the media, five strains were selected and compared to growth curves and cell weights. Four species and Candida albicans A total of five species, including one species, were selected. Among them, one species of Ya was used for this experiment in Saccharomyces cerevisiae.

Example 2. Organic Conversion of Yeast Selected Strains against Selenium and Iodine

Ya, 500 ppm of selenium (added as sodium selenite) and iodine (added with potassium iodine) to saccharomyces cerevisiae, the strain selected in Example 1 of yeast, 500, 1000 Incubated for 24 hours in YM medium added at a concentration of 1500 and 2000 ppm, the cell weight and the concentration of selenium and iodine were measured, and the organic conversion of selenium was evaluated using MBRT.

As a result, as can be seen in Table 1, the dry mater basis is 1.65, 2.11, 2.18 and 1.87% when the iodine concentration is 500, 1000, 1500 and 2000 ppm, respectively, the highest result at 1500 ppm (P <0.05), the amount of iodine in yeast cells was 3509 ppm at 500 ppm treatment, which was significantly higher than other treatment concentrations (P <0.05).

MBRT, an organic conversion rate of selenium present in yeast cells, was 1420, 1657, 1890, and 2022 at 500, 1000, 1500, and 2000 ppm, respectively, and increased with increasing iodine concentration.

Iodine concentration (ppm) Cell weight (%) Iodine (ppm) MBRT (seconds) 500 1.65 ^C ± 0.02 3509.09 ± 9.000 1420.3 ± 38.44 1000 2.11 ^A ± 0.06 3347.00 ± 337.00 1657.5 ± 34.92 1500 2.18 ^A ± 0.02 1813.00 ± 173.00 1890.7 ± 33.67 2000 1.87 ^B ± 0.08 2890.00 ± 10.00 2022.2 ± 3.67 * The mean is significantly different (P <0.05) from the same superscript in the same column as the mean ± SE of the three replicate measurements.

Example 3. Investigation of safety and absorption rate of manufactured organic yeast selenium

In order to investigate the safety and absorption efficiency of yeast selenium prepared using a pilot-scale fermenter in rats and inorganic selenium, the following experiment was performed.

First, 20 SD male rats at 5 weeks of age were exposed and 10 days of acclimatization in individual cages of environmentally controlled metabolic chambers set at a temperature of 22 ± 2 ° C, relative humidity of 50-60%, and a 12-hour contrast cycle. Through the three days of the experiment was published, and the experimental feed and water to be ingested freely. The animals were randomly placed in two treatment groups of 10 animals, and organic selenium was used as yeast selenium, and inorganic selenium was used as sodium selenite. Rats in each treatment group were orally administered 1 ml daily so that the concentrations of organic and inorganic selenium were 2 mg / kg based on dietary intake. Feed intake, weight and excreta of rats were measured daily during the experimental period. Excretion was dried for 3 days at 40 ℃ finely ground and used for selenium analysis.

As a result, the daily gain was 5.1 g / day in the yeast selenium feeding group and 5.3 g / day in the sodium selenite feeding group, as shown in Table 2 below. This weight gain was not significantly different from the standard weight gain in the SD rats reported in this study. Yeast selenium prepared in this study was evaluated as safe although it was evaluated in a short period of time.

As a result of comparing the selenium absorption efficiency with inorganic selenium, the selenium absorption rate of yeast selenium was slightly higher as the absorption rate of Se of yeast selenium was 79.16% and the selenium absorption rate of sodium selenium was 78.72%. There was no significant higher than the absorption rate of selenium of inorganic sodium selenite.

Aid Weight (g) Early End increase Increase / day Yeast Selenium 171.0 ± 1.5 227.0 ± 8.8 56.0 ± 8.0 5.1 ± 0.7 Selenite sodium 170.3 ± 1.5 228.9 ± 5.0 58.6 ± 4.8 5.3 ± 0.4

Item Yeast Selenium Selenite sodium Selenium absorption rate (%) 79.16 ± 0.96 78.12 ± 1.06

Example 4 Development of Cultivation Process at Pilot-scale

4-1. Use strains and culture methods in oil culture

The strain used in this experiment is Saccharomyces cerevisiae Ya strain selected in Example 1. The medium used for the culture was YMP medium as the base medium in the spawn culture, and the medium of the present culture was a modified medium (hereinafter, referred to as GY medium). Each medium composition was 10 g / l glucose, 3 g / l yeast extract, 3 g / l malt extract, 3 g / l peptone, 15.0 g / l glucose, 6.0 g / l yeast extract, magnesium sulfate (MgSO ₄ ) 0.5 g / l, ammonium sulphate ((NH ₄ ) ₂ SO ₄ 3.0 g / l, dipotassium phosphate (K ₂ HPO ₄ ) 1.5 g / l The composition of the feeding medium in the incubation is 400.0 g of glucose / l, yeast extract 40.0 g / l, magnesium sulfate 6.0 g / l, ammonium sulfate 40.0 g / l, dipotassium phosphate 16.0 g / l.

Seed cultures were inoculated with 10 ml of YMP medium in a 35 ml test tube and inoculated with colonies on an agar plate.These were incubated at 30 ° C and 150 rpm in a shaker (Jeio tech, SI-600R). The cells were incubated for 5 hours (V / V) inoculated into a 500 ml Erlenmeyer flask containing 100 ml of YMP medium and shaken for 12 hours at 30 ° C and 150 rpm to be used as the inoculum.

This culture was performed separately in 5 L, 500 L and 5000 L-fermenters.

In case of 5 L culture, put 2 L GY medium into 5 L fermentation tank and inoculate 5% of the above inoculum, and adjust pH 5.5, 30 ℃, aeration volume 1-2 vvm and stirring speed at 300-800 rpm. It was.

In the case of 500 ℓ culture, the temperature, pH and aeration amount were used under the same conditions as the 5 ℓ fermentation tank, and the stirring speed was adjusted to 100 to 300 rpm and 50 to 170 rpm, respectively. A 50 L fermenter (150 rpm, 14 hours) 10% of the culture was used.

Cell mass measurement The cell mass in the culture was measured at 600 nm using a spectrophotometer. The dry cell weight from the absorbance was converted into the absorbance value.₆₀₀Calculated by multiplying. glucose As a result, the glucose remaining in the culture medium was centrifuged at 4 ° C., 8000 rpm, and 30 minutes with a freezing centrifuge from the culture medium sample. Organic selenium was measured by decomposing all organic selenium into selenium in dried cells and measuring the amount of selenium. The organic selenium was measured by a fluorescence spectrometer using a fluorescence assay. This analysis was commissioned by a scientific and technical analysis center.

4-2. Basic experiment in fermenter culture

In order to determine the production medium in a 5 L fermenter culture, the YM medium used in the flask culture of Example 4-1 and generally Saccharomyces cerevisiae were used. GY medium, a fermentation tank production medium, was used for 14 hours batch culture in a fermenter containing 2 liters of medium, and the growth of cells was compared and examined. Optimal feeding medium, i.e. medium containing 0.5 g / l selenium, 0.17 g / l yeast extract, 0.67 g / l sodium sulfate and 11.25 g / l glucose, was added to the culture at 14 hours for 5 hours. Proliferation was investigated. At this time, iodine was added to the initial culture medium to 0.5 g / l. On the other hand, in order to investigate the possibility of oil culture, the change in the concentration of glucose as a carbon source, that is, the change in cell mass in batch culture according to 1.5 g / l, 3 g / l and 15 g / l, was examined for 11 hours.

The growth of cells in each culture was examined using YM medium and GY medium in a 5 L fermentation tank culture, and the results are shown in FIG. 1.

As shown in FIG. 1, after 12 hours of culture, the cell mass in the GY medium and the YM medium was 7 g / L and 3.5 g / L, respectively, and the cell growth in the GY medium was about 2 times higher than in the YM medium.

In addition, the growth of the cells was confirmed by adding an optimum feeding medium, that is, a medium containing selenium, glucose, yeast extract, and sodium sulfate in a 5 L-fermentation broth. It was added at 14 hours of culture, when the carbon source was depleted. As a result, cell growth was not increased due to the addition of oily medium, but the growth of the cells was kept constant, and the same pattern was observed in the YM and GY medium.

As a result, in the case of using GY medium rather than YM medium, the growth of the cells was not only excellent and the effect of the addition of selenium medium was the same.

On the other hand, the change in cell mass in the batch culture according to the change in the concentration of glucose (1.5 g / L, 3 g / L, 15 g / L) as a carbon source in a 5 L fermentation tank was examined, and the results are shown in FIG.

As shown in FIG. 2, the cell mass increased with increasing glucose concentration. However, in the 15% glucose, the lag time was increased as the amount of glucose in the early stage of culture increased, and the cell weight was only about 2 times higher than that of 1.5% glucose. It tended to decrease. This occurs because a large amount of glucose in the early stage of culture inhibits cell growth. The same results as in the fermenter culture. Therefore, in order to obtain an optimal cell mass, fed-batch was evaluated as being desirable.

4-3. Investigation of Oil Price Condition and Maximum Production of Cells

Based on the result of the flask culture of Example 4-2, in order to efficiently obtain cells containing complex organic minerals, selenium, glucose, yeast extract and sodium sulfate were 0.5 g / L and 11.25 g /, respectively, after 24 hours of culture. The conditions for culturing for 24 hours after addition of the medium prepared to contain l, 0.17 g / l and 0.67 g / l were set to optimum conditions. Therefore, as a strategy for mass production of complex organic mineral cells in milk ash, first, two methods of adding medium according to yeast growth under the condition of milk culture, that is, exponential fed-batch culture and intermittent After reviewing the optimal oil price method for the maximum cell production under the condition of intermittent fed-batch culture, the maximum cell mass was obtained under the selected batch condition. Secondly, the cells obtained after culturing for 24 hours by adding selenium to the culture time at which the maximum cell weight was obtained were determined to be recovered.

The exponential oil value cultivation is a method of controlling the supply of the medium by obtaining the microbial cell yield for the carbon source at a specific growth rate (1 / h) of the microorganism from the time when the carbon source is depleted in the medium. personal computer-controlled exponetial feeding method). Fed-batch (F) was examined by supplying the oil-fed medium using the following equation 1 with the specific growth rate (μ, 1 / h) of the microorganisms as 0.15, 0.2 and 0.25, respectively.

Oil (F) = (μ · V 0 · X 0 / Y · S F) exp (μ · t)

μ = specific growth rate (μ, 1 / h)

V ₀ , = initial fermentation broth volume (ℓ)

X ₀ , = initial cell (cell) concentration (g / ℓ)

Y = cell (cell) yield for glucose (g-cell / g-glucose)

S _F = feeding glucose concentration

In exponential fed-batch cultures, the oil price is the on-line data value of the CO ₂ analyzer attached to the culture fermenter, ie the progress of the culture, when the glucose depleted by the yeast is depleted during the culture. As a signal that the measured CO ₂ value rapidly decreased, oil price culture was started. For both types of fed-batch cultures, the culture was started with 2 L of the initial medium and the culture was stopped when 1 L of the fed medium was completely consumed.

In the case of intermittent fed batch culture, the pH of the medium increases after the carbon source is depleted in the medium, which causes the acid line to be fed using the acid stag (pH stat). When the pH of the medium is increased after connecting to the acid line, a certain amount of fed oil medium is fed into the fermenter to carry out the oil value culture.

The exponentially fed-value cultivation in a 5 L jar fermenter was performed by adjusting the specific growth rate (μ, 1 / h) values to 0.15, 0.2 and 0.25, respectively. 3b and 3c.

As shown in FIG. 3a, glucose was consumed after 5 hours of culture, and the cell weight was 3.5 g / l and ethanol was 4.5 g / l. Therefore, feeding began at 5 hours of culture, and cell growth was different at the beginning of oil price and at the end of oil price. That is, the value of μ was adjusted to 0.15 and 0.060 as branching points for 14 hours of cultivation, and the oil price was completed at 24 hours of culturing to obtain 29.4 g / L of cell mass. During the cultivation, ethanol produced 4.5 g / l at 5 hours of cultivation and there was no change, but was gradually produced from 16 hours of cultivation, and 8 g / l at 24 hours of cultivation. It was shown that the cells consumed glucose after 16 hours of culture to produce not only the cells but also ethanol, and the ethanol accumulated in the culture medium affected the growth of the cells and decreased the μ value later in the culture.

FIG. 3b shows the result of controlling the μ value to 0.2. The μ value was adjusted to 0.2 and 0.066 as branching points for 14 hours of cultivation, and the oil value was completed at 17.5 hours of cultivation to obtain 23.14 g / L of cell mass. . During the cultivation, the ethanol proceeded slowly from the beginning of the culture to 6 g / L at 14 hours of culture, and rapidly produced after 14 hours of culture.

3c shows the result of adjusting the mu value to 0.25, and the mu value was adjusted to 0.25 and 0.1 at the branching point for 12 hours of cultivation, and the oil value was completed at 16 hours of culturing to obtain 24.6 g / l of the cell mass. During the cultivation, the ethanol proceeded slowly from the beginning of culture to 6 g / l at 12 hours of cultivation, and rapidly produced after 14 hours to 19 g / l. Taken together, the value of μ decreases rapidly during the middle of oil price because glucose produces ethanol. The higher the value of μ, the more the cell mass increases but ethanol is also produced. Cell growth was inhibited by ethanol. This phenomenon occurs in other Saccharomyces cerevisiae strain culture, it is known that cell growth is inhibited by ethanol of 5 g / L or more in the medium, and also inhibits ethanol production at a concentration of 10 kg / L or more. However, the strain used in this experiment is a strain that produces ethanol up to 19 g / l when the μ value is 0.25, and it may be desirable to cultivate the ethanol while reducing the μ value. However, there is a problem in that the low μ value leads to a decrease in productivity due to a long total oil time.

Therefore, the intermittent oil cultivation of adding glucose when the carbon source was depleted during the culturing was performed, and the results are shown in FIG. 4.

The oil price started at 8 hours of cultivation, which means that glucose was depleted at 5 hours of cultivation but 4.5 g / l of ethanol was present in the medium. Because it was oil price. As can be seen in Figure 6, after the start of the oil price, the μ value was adjusted to 0.2 and 0.1 as the branching point for 14 hours of cultivation, the oil price was completed at 35 hours of culture to obtain a cell weight of 50 g / l. During the cultivation, ethanol produced less than 4.5 g / l after the start of oil price.

In order to evaluate the results of the fed-batch, the correlation of process variables in each condition is shown in Table 4.

As shown in Table 4, in the exponentially fed-batch culture, the lower the value of μ, the higher the cell yield and the maximum cell mass, but the longer the feeding time, the higher the glucose consumption rate (Qs, glucose / g cell / h). I couldn't get it. This is because exponential oil culture can not decrease the Qs value even when adjusted to a low μ value, so that the maximum cell mass Xm cannot be increased. The intermittent oil culture resulted in the highest glucose utilization with a Qs value of 0.081 and the highest value of Xm and Y x / s.

Specific growth rate μ (1 / h) Incubation time t (h) Cell yield Yx / s (-) Max Cell Concentration Xm (g / ℓ) Cell productivity Qp (g / ℓ / h) Glucose Consumption Rate Qs (g / g / h) 0.15 24 0.2 29.4 1.17 0.20 0.2 17.5 0.16 23 1.28 0.35 0.25 16 0.17 24.6 1.49 0.36 Intermittent oil prices 35 0.34 50 1.36 0.08

4-4. Addition of Iodine and Selenium in Fermentor Cultures

The fermentation was carried out in a 5 L jar fermenter with intermittent oily culture, the maximum cell weight production condition of Example 4-3. Iodine proceeded for 35 hours by adding potassium iodine (potassium iodine) to YMP medium to 0.5 g / L at the beginning of the culture. Selenium consumes 0.05 g / l of sodium selenite in a 2 l culture medium after 3 hours to consume the ethanol remaining after 35 hours of intermittent fed-batch culture to starve the cells. Yeast extract and sodium sulfate were incubated for 24 hours with the addition of 300 ml of fed-value medium containing 0.5 g / l, 11.25 g / l, 0.17 g / l and 0.67 g / l, respectively, and the results are shown in FIG.

As can be seen in Figure 5, after addition of potassium iodine and sodium selenite showed a slight increase in cell weight up to 4 hours of incubation, but after that time the cell mass remained constant. This resulted in 52 g / l of cells without a decrease in cell weight after 24 hours of cultivation because not only the minimum amount of glucose and yeast extracts were present in the feeding medium but also selenium acted as an energy source for some cell growth. The obtained culture solution was recovered by freezing and drying the cell concentrate by the method of separating the cells of Example 4-6 later, the amount of selenium and iodine in the dry powder was measured according to the method of Reference Examples 2 to 3, 8147 ppm and 4380 It was confirmed that the ppm contained.

4-5. Cell mass production at pilot scale

In order to realize the production of microorganisms containing complex organic minerals in 500 L and 5000 L fermentation tanks, intermittent oily cultures were carried out, and process variables for each culture were compared. One result is shown.

As can be seen in Figure 6, the oil price began at 10 hours of cultivation, and the L value was adjusted to 0.2 and 0.6, respectively, with the branching point at 14 hours of cultivation. The fed-batch medium was exhausted at 42 hours of culture, and the cell mass at this time was 45 g / l.

In addition, the process parameters of each culture were obtained from the results of the intermittent oil culture, 5 L, 500 L and 5000 L, and are shown in Table 5 below.

As can be seen from Table 5, the total incubation times at 5 L, 500 L and 5000 L were 35 h, 42 h and 43 h, respectively, with the total incubation time at 500 L incubation longer compared to 5 L incubation. It was longer, and there was no significant difference from 5000 ℓ, which also affected Y x / s, Xm, Qp and Qs, resulting in lowering to 5000 ℓ. This result is a phenomenon that occurs during the scale-up process due to the effects of rotational speed and air supply.

Fermentation type Incubation time t (h) Cell yield Yx / s (-) Max Cell Concentration Xm (g / ℓ) Cell productivity Qp (g / ℓ / h) Glucose Consumption Rate Qs (g / g / h) 5 ℓ 35 0.34 50 1.36 0.080 500 ℓ 42 0.31 45 1.03 0.074 5000 ℓ 43 0.31 44.3 1.01 0.073

On the other hand, each culture was obtained in the dry powder by the method of Example 4-6 to measure the selenium content and iodine in the dry powder in order to predict the organic selenium content is shown in Table 6 below.

As can be seen in Table 6 below, the selenium content at 5 L, 500 L and 5000 L was 8147 ppm, 7527 ppm and 6082 ppm respectively and the iodine content was 4380 ppm, 3908 ppm and 3600 ppm respectively. The selenium and iodine contents showed lower values as the fermentor scale became larger.

Fermentation type Selenium Concentration in Dry Powder (ppm) Iodine Concentration in Dry Powder (ppm) 5 ℓ 8147 4380 500 ℓ 7527 3908 5000 ℓ 6082 3600

4-6. Concentration and Drying of Cells Containing Complex Organic Minerals

In order to obtain a composite organic mineral dry matter, a process of concentrating and drying the cells from the fermentation broth was developed. The concentration and drying of the cells from the 3000 L culture solution obtained after the incubation in a 5000 L fermentation bath were performed by mixing the Pall-sep (pall sep VMF 400 system), the dispersion medium, which is a vibration membrane filter connected to the 5000 L fermentation tank. It was carried out using a cell crusher (SPM-15) and a lyophilizer used as (beads), and the yield was evaluated. The flow chart of the concentration of the cells and the preparation of dry powder is shown in FIG.

As shown in FIG. 7, Pall-sep (pallsep VMF 400system), a vibration membrane filter connected to a 5000L fermentation tank, was used to separate the cells from the 3000L culture solution obtained after the incubation in a 5000L fermentation tank. It was. The cell concentrates obtained from the Pall-sep 0.45 μm membrane filter were recovered in a 5000 L fermenter and 600 L of concentrated culture was obtained, which took 4 hours. In order to wash the obtained cells, 1200 L of water was added to a 5000 L fermenter and passed through Pall-sep to obtain 600 L of cell concentrate. The required time was 4 hours. This washing process was repeated twice, the last washing process was concentrated to 300 L and took 5 hours. The concentrated 300 L culture solution was disrupted by the cell crusher in the following process. At this time, the working volume of the cell crusher was 16.9 L, and it took 3 hours to crush 300 L by processing 100 L concentrate per hour in a continuous process at 100 rpm. The crushed concentrate was dried for 3 days in a 300 L freeze dryer to obtain 138 kg of cells.

That is, as shown in Table 7, the cell mass obtained after concentration and freeze-drying from the 3000 L culture solution was 138 kg, yield was 4.3%, and the recovery amount compared to the dry cell weight of the culture medium was 90%.

Fermentation broth Total dry cells in fermentation broth Dry powder obtained from freeze drying Recovery per fermentation broth Recovery rate from fermentation broth 3000 ℓ 145 130 4.3 90

The organic selenium-iodine complex using yeast prepared according to the present invention not only prevents various diseases, but also has no toxicity to the human body, and has a high absorption rate, so that it may be used as a feed additive, a health functional food, or a medicine.

Claims (14)

1 in my process serenity busy as Saccharomyces (Saccharomyces cerevisiae) or Candida albicans step of culturing the microorganisms of (Candia albicans), (2) the My process serenity busy as Saccharomyces (Saccharomyces cerevisiae) or Candida albicans (Candia To incubate the spawn of albicans ), two steps of inoculating and cultivating in an iodine-added medium, (3) three hours of cultivating an optimum milk medium containing selenium after incubation for a predetermined time, and (4) a complex Organic selenium-iodine comprising four steps of concentration, cell disruption and drying of Saccharomyces cerevisiae or Candida albicans cells containing selenium and iodine complexes as organic minerals. Method for preparing a composite. delete According to claim 1, wherein the Saccharomyces cerevisiae ( Saccharomyces cerevisiae ) is Saccharomyces cerevisiae ( Saccharomyces cerevisiae ) Ya (KCTC7107, sold at the Korea Biotechnology Institute Gene Center), Saccharomyces cerevisiae Saccharomyces cerevisiae Yb (KCTC7919, sold at Korea Biotechnology Research Institute Genetic Center), Saccharomyces cerevisiae ) Yc (KCTC7928, sold at Korea Biotechnology Research Institute Genetic Center), Saccharomyces cerevisiae Saccharomyces cerevisiae Yd (KCTC7965, sold at the Korea Biotechnology Research Institute Gene Center) or Saccharomyces cerevisiae Ye (KCTC7963, sold at the Korea Biotechnology Research Institute Gene Center). The method of claim 3, wherein ( Saccharomyces cerevisiae ) is Saccharomyces cerevisiae ( Saccharomyces cerevisiae ) Ya (KCTC7107, sold at the Korea Biotechnology Research Institute Gene Center). The method of claim 1, wherein the medium used in the second and third steps is GY medium. The method of claim 1, wherein the iodine of the second step is 0.1 to 1.0. g / l added. The method of claim 1, wherein the feed concentration of selenium in the oil medium used in the third step is 50 to 6000 ppm. 8. A process according to claim 7, wherein the feed concentration of selenium is 500 ppm. The method of claim 1, wherein the oil-containing medium containing the selenium of the third step is a constituent of the medium, in addition to selenium, glucose, yeast extract and sodium sulfate are additionally included. 2. The process according to claim 1, wherein the source of selenium in the third step uses all compounds containing selenium and selenium as inorganic selenium. The method of claim 10 wherein the selenium source is sodium selenite. The production method according to claim 1, wherein the type of culture in the third step is intermittent oily culture. The method according to claim 1, wherein the selenium-containing oil medium used in the third step is added after 20 to 40 hours of incubation, and further cultured for 20 to 30 hours after the addition of the selenium-containing oil medium. Way. The method of claim 1, wherein in the fourth step, the cells containing the complex organic minerals are concentrated using a membrane separation filtration device, lyophilized using a cell crusher and a lyophilizer.