HU205379B - Process for producing yeasts enriched with microelements - Google Patents

Abstract

Candida utilis and saccharomyces cerevisiae yeasts are enriched by micro elements when these are added to the fermentation medium. The microelements enter into the yeasts as chemical cpds. The biotransformed elements show advantageous activity and may be used in fodder, foodstuffs, pharmaceutical prods. and in cosmetic

Description

FIELD OF THE INVENTION The present invention relates to a process for the preparation of micro-nutrients enriched with trace elements, preferably iron, copper, zinc, manganese, molybdenum, cobalt, vanadium, lithium, arsenic, silicon, titanium, tellurium, nickel, cesium, rubidium, iodine or fluorine, in the form of their salts in the medium or added during the growth of the fungi.

The added micronutrients are enriched intracellularly in fungi, incorporated into a large proportion of organic compounds, and as new types of micronutrient sources are more suitable and advantageous for administration to human and animal organisms.

In practice, there are 65 known microelements that have or may have physiological effects (András Szabó S. et al., Microelements in Agriculture I, Agricultural Publisher, Budapest, 1987, and István Pais: The Importance of Micronutrients in Life, University of Horticulture and Food, Budapest, 1989)

Trace elements, as it is known, exert their physiological effects by being incorporated into the organic molecule, structural and enzyme proteins, or by their presence activating the enzyme. The body needs a certain amount of micronutrients and the metabolic losses need to be compensated continuously. In addition to food and drinking water, the supply of trace elements is provided by the organic and / or inorganic salts of the trace elements. Due to their unfavorable absorption, distribution and depletion, micronutrient supply is still not a problem today, for example, we refer to the most common diseases caused by iron and fluoride deficiency.

For example, U.S. Patent No. 3,345,211 is known from the patent literature. A disclosure document in the Federal Republic of Germany stating that yeast enriched in germanium is produced. No. 4,348,483. U.S. Pat. No. 4,530,846; U.S. Pat.

Thus, it is known that micronutrients play a very important role in medicine and prevention, and some patents are known which provide solutions for the production of yeast rich in micronutrients. However, there is a need for more human and animal organisms It is also possible to prepare a micronutrient which is important for it, in the form of a physiologically configurable, novel type of biotransformed micronutrient source.

Accordingly, it is an object of the present invention to provide a process that can meet this need.

It is well known that certain microelements in the form of organic salts are toxic, but in their natural form are harmful to the body. Therefore, in accordance with the invention, further aspects that are decisive for the fermentation process have been determined. to accomplish this process with respect to the particular micronutrients provided, so as to achieve the object outlined above. We have discovered that yeast can be further enriched in the micronutrients we label, provided that the process parameters are determined in advance.

The method of the present invention is based on the mechanism by which the micronutrients added during the growth of yeasts are enriched intracellularly and are organically bound. The yeasts are grown in a known manner and in a known medium. The water-soluble salts of the trace elements are weighed into the medium or added during growth. The addition is carried out simultaneously, in several portions or continuously, depending on the salt of the trace element and the yeast. The amount and nature of the salt added is determined by the effect of the particular salt of the micronutrient on reproduction. Inhibitory salts are added in low concentrations and in several portions. The amount of salt added and the mode and time of administration are determined by monitoring growth and micronutrient concentration. This should be stated separately for each trace element salt. The conditions of administration are determined so that they do not result in a significant reduction in the yeast cell mass and sufficient enrichment. After growth, the cell mass is separated, washed with water, filtered and dried if desired.

The patent literature, as already mentioned, discloses three methods for enrichment of microelements. U.S. Pat. No. 4,348,483 discloses the production of chromium-enriched yeast by the addition of chromium chloride with about 1000-4000 pg Cr / g of dry yeast fortification. The enrichment was performed in Saccharomyces cerevisiae and the salt was added at the end of growth. U.S. Patent 4,530,846 describes the preparation of selenium-enriched yeast. Sodium selenite was used for enrichment, which was continuously added to the growth cycle and reached 14402000 pg / g of selenium enrichment in Saccharomyces cerevisiae and S. uvarum. German Patent No. 3,345,211 relates to the production of yeast enriched with gennanine. By the addition of germanium citrate, germanium enrichment of 400-700 pg / g was achieved in several types of yeast (S. cerevlsiae, S. carlsbergensis, Torulopsis utilis).

In accordance with the present invention, a method of enriching an additional 17 micronutrients has been developed, with each micronutrient or salt being individually identified for enrichment. In the process of the invention, the following parameters are determined:

(a) whether the accumulated salt has an inhibitory effect on the growth of yeast:

(b) at what concentrations the salt does not reduce yeast growth;

(c) the manner in which the salt is to be administered (in one, several, continuous);

d) at which stage of the growth the particular salt is to be added to the culture medium;

(e) the effect of the salt on the processing of biomass;

(f) the extent to which the micronutrient input by the addition of a given salt is capable of accumulating in yeast.

In the product of the present invention, where organic bonded biotransformed microelements are present, they have a better absorption coefficient, reducing inorganic microorganisms.

EN 205 379 Β elements, they are less toxic, have a pleasant taste, can be positively dosed and can be formulated more easily. The chemical composition of the yeast also contributes to the beneficial effect. Saccharomyces cerevisiae (baker's yeast) has a cell weight of 40-50% protein. The most abundant vitamins are thiamine 390, riboflavin 2200, niacin 21,000, pyridoxine 760, pantothenic acid 12,000 and folic acid at 170 pg / 100g. By enriching the micronutrients in yeast, we obtain a source of naturally occurring micronutrients.

The process of the present invention utilizes the recognition that yeasts are capable, under certain conditions, of enriching certain microelements intracellularly, up to thousands of times their normal state, and of bringing them into organic binding.

The following Table 1 lists the concentrations of the 17 micronutrients we investigate in the conventional, self-known baker's yeast, which is produced by molasses in the known industrial process, and the titers of the micronutrient-enriched yeasts we produce from the same elements. Mean values for normal baker's yeast are based on literature data and our own studies (Rodney, P.J. and Greenfield, P.F., Process Biochemistry, 1984, 4, 4860).

Table 1

microelement The micronutrient concentration is µg / g normal yeast enriched yeast Fe 80 1000-10,000 Cu 27 500-1000 Zn 150 500-5000 Mn 20 300-1200 Mo 4.04 2000-12,000 Co 3 200-500 V 0.05 1000-1500 Li <0.01 10-100 Dig 1 500-1000 Ski 30 1000-6000 You 0.1 2000-12,000 You <1 1000-2200 Ni 1 100-300 Thurs <1 50-200 rb <1 100-400 11 20 80 F 1 50-100

Another important feature of yeast fortified micronutrients is that they contain most of the accumulated micronutrient in organic form. To illustrate this, we present Table 2, which shows the results for iron, listing all the iron in the organic bond and the iron in the organic bond. It can be seen that the incorporation into the organic bond is always greater than 90%.

Table 2

Yeast Fe uptake (Pg / g) Integrated Fe (pg / g) incorporation (%) Saccharomyces cerevisiae 7300 7100 97 Saccharomyces cerevisiae 3800 3470 '91 Saccharomyces cerevisiae 3400 3220 95 Saccharomyces cerevisiae 8900 8700 98 Candida utilis 2300 2260 98 Candida utilis 1100 1015 92

The process of the present invention has been carried out with baker's yeast Saccharomyces cerevisiae, which is also suitable for human consumption, and with a feed yeast Candida utilis, but it is also applicable to other yeasts. Both strains were maintained on malt medium containing 5% malt juice and 2.5% Bacto agar. After inoculation, the strains were incubated for 48 hours at 30 ° C and stored in a refrigerator until use (24-48 hours).

Yeast propagation and accumulation of trace elements were performed in shake flasks and in a fermentor. Shake cultures were also inoculum for fermenters.

Culture composition of the shake flask culture: glucose 50.0 g / dm ³ (NH ₄ ) ₂ SO ₄ 5.0 g / dm ³

KH ₂ PO ₄ 1.0 g / dm ³ yeast train 3.0 g / dm ³ desertobiotin 0.04 mg / dm ³ pH 4.5 (with 15% HCl)

The flask medium was sterilized in an autoclave at 1.2 bar for 15 minutes. Sterile medium was 100 to 100 cm ³ of 750 nm in ^three inch Erlenmeyerlombikokban inoculated agar culture tubes are washed in sterile water 1-1 csövéről yeast slurry. Propagation was carried out on a 350 rpm shaker at 30 ° C for 20-24 hours for both inoculum and flask series. Flask series were inoculated with a previously started flask inoculum at 10%. The micronutrient salt was added at the inoculation from a previously prepared stock solution to the uninoculated medium.

For fermentation growth, 10 dm ³ fermenters with 5 dm ³ medium were used.

glucose 70.0 g / dm ³ (NH ₄ ) ₂ SO ₄ 7.0 g / dm ³

KH ₂ PO ₄ 1.5 g / dm ³ yeast extract 5.5 g / dm ³ desertobiotin 0.06 mg / dm ³ pH 4.5 (with 15% HCl)

HU 205 379 Β

In Feimentor culture, the glucose components were sterilized separately in a flask to avoid undesired staring (Maillard reaction) and placed in the fermentor prior to inoculation. Again, the inoculum was 10% flask culture. Aeration of 1 dm ³ / min / dm ³ and stirring at 700 rpm was used for 24 h propagation. The growth temperature was 30 ° C. The salts of the trace elements were added during the propagation in the amounts and in the manner described in the examples.

At the end of the propagation, the yeast mass was separated from the growth medium by centrifugation for 30 minutes at 3000 rpm. After washing three times with tap water and repeated centrifugation, excess acetone was added to the pulp, causing the biomass to be punctured as fine flakes. After filtration, the yeast was dried at room temperature. The aforementioned acetone treatment allowed complete removal of the remaining extracellular trace element from the cell surface after washing three times. However, some of the intracellular inorganic-bound micronutrient also came out of the cell due to the destruction of the plasma membrane. Cell lysis experiments showed that the cell mass obtained above contained at least 90% of the enriched trace element in organic binding, as shown in Table 2 for iron.

During growth, optical density values (OD), changes in micronutrient concentration, glucose depletion and pH were measured.

The change in yeast cell mass (OD) was monitored by measuring cell density. Our samples were measured at 10 nm dilution spectrophotometrically at 550 nm against a similar dilution of uninoculated medium. For dilution, 0.01 N HCl solution was used.

Trace element concentrations were measured from the culture medium, the dried yeast samples and the filtrate of the digested samples. The various micronutrients were determined on a LABTEST (Los Angeles, Califomia) PLASMALAB by ICP-AES.

Sample preparation for ICP measurement:

0.5 g of the yeast samples were weighed into a special acid-resistant Teflon bomb for destruction. The sample was 3 cm ³ cc. The mixture was left standing at room temperature overnight with HNCl2 + 3 cm ³ H2O2 and then heated at 100 ° C for the next 30 minutes. After cooling, the solution was made up to 10 cm ^{3 with} 2 times distilled water for analysis.

The micronutrient concentration of the fermentation broths was measured immediately after removing the cell mass by centrifugation.

Measurement was performed using the LABTEST PLASMALAB Inductively Coupled Plasma Atomic Emission (ICP-AES) method.

Circumstances in which required:

radio frequency power: 1.3 kW external argon flow rate: 12 dm ³ / min intermediate argon rate: 0.8 dm ³ / min internal argon pressure: 300 kPa sample rate: 2.9 cm ³ / min

Signal and data processing with digital integrators per channel, respectively. using special calibration and measurement programs running on a computer

In the case of halogens (I, F), micronutrient concentrations were measured with non-selective electrodes.

The concentration of iodine in the samples was measured by direct potentiometric method using a calibration curve using a RADELKIS iodide selective electrode and a saturated cross-linking electrode. Measurement of iodine content of biomass was preceded by destruction according to methods developed by AOAC Methods

Weigh 0.1 to 0.5 g of the sample into a nickel crucible and suspend in 5 cm ^{3 of} saturated alcoholic KOH. Dry in a sand bath, gently charcoal on flame and incinerate at 600-650 ° C for 3 hours. After cooling, the ash is slurried with water and transferred to a 50 cm ³ volumetric flask. Neutralize with HNCL (1: 1) while bubbling. Then, with 600 g / dm ³ K-acetate solution, the pH of the solution was adjusted to ca. Set to 5 and make up to 50 cm ³ with water.

Like iodine, fluorine was measured by a potentiometric method using a selective electrode. Prior to measuring the fluorine content, the dried biomass was subjected to the following digestion:

In a nickel crucible, weigh 0,1 to 0,5 g of the sample to be determined and 0,1 g of CaO. (CaO is needed to fix the fluorine.) Suspend in a little water, dry in an asbestos mesh, gently charcoal on flame, and incinerate at 600-650 ° C for 2 hours. There are two ways to process it further:

a) After cooling to ash, 3 g of NaOH are added and the furnace is melted at 600 ° C for 1 hour. After cooling, the portionwise addition of a total of 38 cm ³ 1 mol / dm ³ citric acid is suspended or dissolved. Finally, 50 cm ³ volumetric flask filled up to 50 cm ^3rd

b) Cremation is followed by microdiffusion; wash the ash with 2 cm ^{3 of} water into a diffusion vessel and add 2 cm ^{3 of} diffusion acid (2 g Ag 2 SO 4 + 1 cm ³ water + 50 cm ³ + cc HClO 4). At the top of the dish, place a strip of filter paper impregnated with 80 μΐ of 9 g / dm ³ NaOH solution and keep in a 60 ° C oven for 24 hours. After opening, cut the edges of the paper, place the piece of fluorine in a 25 cm ³ beaker, 2 cm ³ water, and 2 cm ³ USAB solution (116.9 g NaCl + 29.5 cm ³ 96% acetic acid + 204, 13 g of sodium acetate »3H2O filled with water to 2000 cm ³⁾ was added. Measure the F 'content without mixing.

To determine the proportion of microelements incorporated into yeast cells by organic binding or only adsorbed, the biomass was subjected to digestion.

A 10-fold dilution of the dried biomass was made with phosphate buffer pH 6.0 and homogenized in a ULTRASONIC homogenizer (Braun, 300 W) for 3 x 3 minutes. The suspension containing the digested cells was stored frozen at -24 ° C until use. Prior to ICP measurement, the thawed samples are VAC

The supernatant was separated from the cellular debris by centrifugation at 30,000 rpm for 30 minutes at a cooled Janetzky centrifuge, then 0.2 cm ^{3 of} 3% trichloroacetic acid was added to 10 cm ^{3 of} solution and the precipitated proteins were filtered through a membrane filter. The birth was analyzed and its micronutrient content indicates the presence of an intracellular, inorganic-bound micronutrient.

The process of the present invention will now be described by way of example 35 with respect to the aforementioned trace element 17.

Execution examples

Example 1

Iron-enriched Saccharomyces cerevisiae

The potential inhibitory effect of the salt used, in this case Fe (II) sulfate, on the growth of yeast was determined by flask growth. The iron concentrations used in the medium were 20.40,100 and 200 mg / dm ³ Fe, respectively. In addition to optical density (OD) values reflecting yeast growth, iron uptake is also shown in Table 3:

Table 3

Media Fe content (mg / dm ³ ) OD recorded Fe / biomass (Pg / g) 0 2.95 - 20 3.05 - 40 3.20 1650 100 3.15 3670 200 2.75 9250

The highest concentration of iron (100 mg / dm ³ ) was used for flask fermentation. Iron (II) sulfate was added simultaneously to the culture medium at the start of the growth, and the growth was done without pH control (Table 4).

Table 4 Time (H) OD Fe conc. in the fermentation broth (mg / dm ³ ) 0 0.45 1.2 2 0.90 4 1.30 37.5 6 2.50 - 8 3.75 39.3 10 5.15 12 5.40 39.5 24 6.25 41.0

Fe intake: 13,000 gg / g dried yeast enrichment: 160 fold

Under the conditions used, FeSCL only dissolved in solution with a gradual decrease in pH, which explains the increase in Fe concentration over time,

Example 2

Iron-enriched Saccharomyces cerevisiae

As in the first example, the potential growth inhibitory effect of iron salt, in this case Fe (1H) citrate, was determined in a flask preliminary. The Fe concentrations used were 25, 50, 125 and 250 mg / dm ^3, respectively. Reproductive OD values for iron uptake are shown in Table 5.

Table 5

Media Fe content (mg / dm ³ ) OD recorded Fe / biomass (Pg / g) 0 3.05 - 25 3.10 - 50 3.25 2120 125 3.20 4270 250 3.20 10,180

Iron uptake increased almost in direct proportion to the amount of iron ingested, but yeast did not inhibit reproduction. For demonstration of fermenter growth, a starting concentration of 100 mg / dm ³ Fe was selected as in the first example. Iron (III) citrate was added in one portion to the medium at the start of the propagation, and no pH control was performed (Table 6).

Table 6

Time (H) OD Fe / fermentation broth (mg / dm ³ ) 0 0.45 80.3 2 0.85 - 4 1.25 74.9 6 2.45 - 8 3.20 71.4 10 5.00 - 12 5.85 64.2 24 6.20 32.1 Fe uptake: 7300 gg / g dried yeast enrichment: 90-fold

As in Example 1, good aeration in the fermentor results in better iron uptake than in flask growth with the same iron concentration.

HU 205 379 Β

Example 3

Iron-enriched Candida utilis

As in Example 1, for Fe (H) sulfate, Fe concentrations of 20, 40, 100, and 200 mg / dm ³ were used to determine the most favorable iron concentration in fodder yeast. Table 7 shows the proliferation OD values and iron uptake

Table 7

Media Fe content (mg / dm ³ ) OD recorded Fe / biomass (Pg / g) 0 9.8 - 20 11.5 - 40 11.2 1820 100 10.9 3580 200 9.0 6990

In the case of feed yeast, iron intake was similar to that observed in baker's yeast and was proportional to the amount of iron intake. Fermentation propagation was carried out as described in Example 1, with 100 mg / dm ³ Fe (© sulfate) added to the culture medium at the start of the propagation. No pH control was performed during the propagation process (Table 8).

Table 8

Time (H) OD Fe / fermentation broth (mg / dm ³ ) 0 1.15 105 2 1.25 - 4 1.65 105 6 3.10 - 8 4.75 93 10 7.20 - 12 10.55 72 24 16,30 16 Fe uptake: 8380 pg / g dried yeast

As in Example I, in a fermenter, good iron uptake results in better iron uptake than in flask growth at the same concentration.

Example 4

Iron-enriched Candida utilis

Fermentation of ferric citrate was also preceded by a flask preliminary experiment. The Fe concentrations used were 25, 50, 125, and 250 mg / dm ^3, respectively. The OD values for growth and the iron uptake are shown in Table 9.

Table 9 Media Fe content (mg / dm ³ ) OD recorded Fe / biomass (Pg / g) 0 8.70 - 25 9.35 - 50 9.70 2150 125 10.05 4230 250 8.85 10,150

For enzyme growth, the procedure described in Example 1 was followed by adding Yas (HI) citrate equivalent to 100 mg / dm ³ Fen at the start of the growth. No pH control during the propagation process (Table 10):

Table 10

Time (H) OD Fe / fermentation broth (mg / dm ³ ) 0 1.10 92 2 1.30 - 4 2.05 83 6 4.25 - 8 6.95 71 10 9.10 - 12 11.75 57 24 15.80 22

Fe uptake: 8800 pg / g dried yeast

Iron enrichments with good aeration as shown in Figures 1-4. are more effective than flask propagations with lower oxygen supply.

Example 5

Iron-enriched Saccharomyces cerevisiae

The propagation described in Example 2 can also be carried out using iron (©) -ammonium citrate as a source of iron. See Table 11 for the latter salt enrichment. The administration is an amount of iron (III) ammonium citrate equivalent to 100 mg / dm ³ Fe added at the beginning of the growth. Propagation was carried out without pH control.

Table 11

Time (H) OD Fe / main menu (mg / dm ³ ) 0 0.25 85 2 0.35 - 4 0.65 69 6 1.30 -

HU 205 379 Β

Time (H) OD Fe / fermentation broth (mg / dm ³ ) 8 3.45 58 10 4.10 - 12 4.25 51 24 4.30 35

Fe uptake: 5100 pg / g dried yeast enrichment: 60 fold

Example 6

Iron Enriched Candida Utilis The propagation described in Example 4 can also be performed using iron (III) ammonium citrate as an iron source. See Table 12 for the latter salt enrichment. The dosage is iron (in) ammonium citrate equivalent to 100 mg / dm ³ Fe added at the beginning of the growth. Propagation was carried out without pH control.

Table 12

Time (H) OD Fe / fermentation broth (mg / dm ³ ) 0 0.80 87 2 1.05 - 4 1.30 70 6 2.35 - 8 7.05 46 10 12.50 21 12 15.00 13 24 26.85 10

Fe uptake: 3600 pg / g dried yeast

Example 7

Copper-enriched Saccharomyces cerevisiae When cultivated in various copper-containing media, baker's yeast was tested for the concentration of copper that inhibits reproduction. Copper was added to the medium as copper (H) sulfate. In flask experiments, the copper concentrations used in the medium were 10, 20.50, 100 and 200 mg / dm ^3, respectively. The effect of ares on the growth of yeast is shown in Table 13.

Media Cu content (mg / dm ³ ) OD recorded Cu / biomass (Ug / g) 100 0.50 X 200 0.45 X x: biomass was not recoverable

Being a good fungicide, copper (II) sulfate, even at low concentrations, strongly inhibited the growth of yeast. Therefore, copper was added to the nutrient medium at 10 mg / dm ³ concentration by fermentation propagation. Table 14 shows the data for a fermentation growth where copper (II) sulfate was added in one portion to the culture medium at the start of the exponential growth phase, without pH control.

Table 14

Time OD Cu / fermentation broth (h) (mg / dm ³ )

0 0.30 2 0.45 - 4 0.60 9.2 6 0.55 - 8 0.65 8.3 10 0.85 - 12 1.40 7.1 24 2.25 6.9

Cu uptake: 1330 pg / g dried yeast enrichment: 50 fold

Example 8

Copper-enriched Saccharomyces cerevisiae Copper inhibited the growth of baker's yeast at a very low concentration (10 mg / dm ³ ) during the propagation shown in Example 7, but the copper uptake was adequate. Therefore, it is expedient to add copper (µ) sulfate to the culture medium in several portions during the exponential phase of propagation as shown in Table 15. No pH control was performed during the propagation, and the salt was added to the broth at 10 mg / dm ³ in 5 divided portions.

Table 13 Media Cu content (mg / dm ³ ) OD recorded Cu / biomass (Fg / g) 0 3.50 10 1.25 1650 20 0.90 X 50 0.55 X

Table 15

Time (H) OD Cu / fermentation broth (mg / dm ³ ) 0 0.30 - 2 0.45 - 4 0.60 1.9 5 0.65 3.6 6 0.95 4.5 7 1.35 5.6

HU 205 379 B

Time (H) OD Cu / fermentation broth (mg / dm ³ ) 8 1.70 7.3 10 2.45 - 12 3.20 6.5 24 4.05 6.0

Cu uptake: 705 pg / g dried yeast enrichment: 26 fold

Continuous dosing does not achieve the same degree of enrichment as single copper intake, but is more favorable in terms of bio mass recovery and optimum copper uptake.

Example 9

Copper enriched Candida utilis

When propagated in various copper-containing media, the feed scavenger was tested for the concentration of copper that inhibits reproduction. Copper was added to the medium as copper (H) sulfate. In flask experiments, the copper concentrations used in the medium were 10, 20 50, 100 and 200 mg / dm, respectively. There were ³ . The effect of copper on yeast growth is shown in Table 16.

Table 16

Media Cu content (mg / dm ³ ) OD Cu / biomass absorbed (pg / g) 0 7.25 - 10 7.50 850 20 7.35 1700 50 7.40 3150 100 7.30 3580 200 2.15 3760

The growth of feed yeast is not significantly inhibited by copper up to a concentration of 100 mg / dm ³ compared to that observed in baker's yeast. Enrichment of copper in yeast, although not commensurate with the amount ingested, is significant.

Fermentation growth is shown at a Cu intake of 100 mg / dm ³ (Table 17). The salt was added in one portion to the medium at the start of the exponential phase, with no pH control.

Table 17

Time OD Cu / fermentation broth (h) (mg / dm ³ )

0.70

0.95

1.05 99

1.85

Time (H) OD Cu / fermentation broth (mg / dm ³ ) 8 4.20 96 10 9.85 - 12 14.75 76 24 17,00 77 Cu uptake: 890 pg / g dried yeast

Example 10

Zinc Enriched Saccharomyces cerevisiae When grown in media containing various concentrations of zinc, the yeast was tested for inhibition of yeast growth by increased zinc content of the medium and the ability of the yeast to accumulate zinc from different concentrations of zinc. Zinc was added to the medium as zinc sulfate. The results of flask propagation are shown in Table 18.

Table 18

Media Zn content (mg / dm ³ ) OD Zn / biomass uptake (pg / g) 0. 3.25 - 10 3.30 750 20 3.20 1250 50 3.20 2730 100 3.15 3760 200 3.10 4550

Enzymatic growth is shown at Zn intake of 50 mg / dm ³ , since from the flask growth data (Table 18), it is clear that above this concentration the zinc accumulation is no longer proportional to the amount administered. A19. The data shown in Table II were obtained from growth without pH control and zinc sulfate was added in one portion at the beginning of the exponential growth phase.

Table 19

Time OD Recorded Zn / fermentation broth (h) (mg / dm ³ )

0 0.30 - 2 0.45 - 4 0.75 55 6 0.90 51 8 1.95 48 10 3.05 - 12 3.50 45 24 3.75 45

Zn uptake: 2050 pg / g plowed yeast enrichment: 14-fold

HU 205 379 Β

Example 11

Zinc enriched Candida utilis

Zinc forage yeast propagation was carried out as described in Example 10. Flask propagation and fermentor biomass production were carried out with the addition of zinc sulfate. The results of the test for the potential inhibitory effect of zinc are summarized in Table 20.

Table 20

Media Zn content (mg / dm ³ ) OD Zn / biomass uptake (pg / g) 0 9.40 - 10 11.30 720 20 11.20 850 50 9.95 1160 100 10.80 2150 200 9.25 4280

Enzyme growth was shown at a concentration of 100 mg / dm ³ Zn. Zinc sulfate was added in one portion to the medium at the start of the exponential growth phase, and no pH control was performed. See Table 21 for our data.

Table 21

Time (H) OD Zn / fermentation broth (mg / dm ³ ) 0 0.65 2 0.85 - 4 1.50 86 6 4.60 76 8 8.50 75 10 9.75 73 12 10.50 73 24 12.55 71 Zn uptake: 660 gg / g dried yeast

Under favorable aeration conditions, the growth of fodder yeast was already more strongly inhibited by zinc sulphate than by flask growth, and the uptake of zinc was also lower.

Example 72

Manganese-enriched Saccharomyces cerevisiae

When cultured in media containing different concentrations of manganese (10,20.50, 100 and 200 mg / dm ³ ), the yeast was tested for inhibition of the growth of the yeast by the increased manganese content of the medium and the amount of manganese in the medium containing can accumulate. Manganese was added in the form of manganese sulfate in the medium. The results of flask propagation are summarized in Table 22.

Table 22

Media Mn content (mg / dm ³ ) OD Intake Mn / biomass (pg / g) 0 3.10 - 10 3.15 45 20 3.20 60 50 3.05 120 100 2.95 185 200 2.20 420

For enzyme growth, 100 mg / dm ³ Mn was chosen because no significant growth inhibition was observed in flask growth, but manganese accumulation was low. Manganese sulfate was added to the culture medium in five portions per hour during the exponential phase of propagation, with no pH control. See Table 23 for our data.

Table 23

Time (H) OD Mn / fermentation broth (mg / dm ³ ) 0 0.35 2 0.45 - 4 0.65 21 5 0.95 45 6 1.25 66 7 1.90 84 8 2.75 105 10 3.15 102 12 3.60 97 24 4.40 95

Mn uptake: 360 gg / g dried yeast enrichment: 18-fold

Example 13

Manganese-enriched Candida utilis

The manganese-enriched feed yeast is prepared as described in Example 12. Flask propagation and fermentor biomass production were performed with the addition of manganese sulfate. Table 24 summarizes the results of manganese inhibitory activity and pre-accumulation.

Table 24

Media Mn content (mg / dm ³⁾ OD Mn / biomass uptake _; (pg / g) ' 0 10.85 10 10.90 145 20 11.40 220 50 11.85 480

HU 205379 Β

Media oldη Content OD Intake Mn / biomass (mg / dm ³ (Pg / g) 100 12.90 1020 200 10.55 1880

Similarly to Example 12, a concentration of 100 mg / dm ³ Mn was chosen for enzyme growth. Preliminary measurements (Table 24) showed the highest bio-mass formation and the favorable accumulation of manganese. The manganese sulfate was added in one portion to the medium at the start of the exponential phase, with no pH control.

Table 25

Time OD Mn / fermentation broth (h) (mg / dm ³ )

0 1.05 - 2 1.15 - 4 1.65 105 6 2.80 105 8 5.85 98 10 13.90 96 12 16.75 93 24 19.50 90

Mn uptake: 1100 pg / g dried yeast

In contrast to baker's yeast, feed yeast has a good agreement between flask and intensive aeration fermentation propagation.

Example 14

Saccharomyces cerevisiae enriched with molybdenum

By growing molybdenum in media containing various concentrations (20, 50, 100 and 200 mg / dm ³ ), the yeast was tested for inhibition of yeast growth by the molybdenum content of the medium and the ability of the yeast to accumulate molybdenum from different molybdenum concentration media. Molybdenum was added to the medium as ammonium molybdenate. The results of flask propagation are summarized in Table 26.

Table 26

Nutrient Mo content (mg / dm ³ ) OD Mo / Biomass uptake (Pg / g) 0 3.05 20 2.30 1650 50 2.40 5570 100 2.55 9520 200 2.45 16230

Baking yeast accumulates molybdenum well during flask propagation with a 20-22% decrease in biomass.

In a fermenter, a concentration of 50 mg / dm ³ Mo is used. ammonium molybdenate was added in one portion at the beginning of the exponential phase of propagation, and no pH control was performed. Table 27 shows our data.

Table 27

Time (H) OD Mo / fermentation broth (mg / dm ³ ) 0 0.35 - 2 0.45 - 3 0.75 52 5 1.15 - 7 2.50 52 9 3.05 51 11 3.25 45 23 3.50 41

Mo-up: 2300 pg / g dried yeast enrichment: 58,000-fold

The molybdenum uptake achieved results in an extra high enrichment compared to the molybdenum content of normal baker's yeast.

Example 15

Molybdenum-enriched Saccharomyces cerevisiae A14. The procedure described in Example 1B was used, except that during the laboratory fermentation, ammonium molybdenate was added in five portions per hour during the exponential phase of growth. Our results are summarized in Table 28.

Table 28

Time (H) OD Mo / fermentation broth (mg / dm ³ ) 0 0.40 - 2 0.45 - 3 0.65 11 4 0.80 23 5 1.45 34 6 1.55 45 7 2.30 51 11 2.75 46 24 3.15 45

Mo intake: 3900 pg / g dried yeast enrichment: 98,000-fold

The addition of ammonium molybdenate in several increments can further enhance the enrichment.

HU 205 379 Β

Example 16

Candida utilis Molybdenum Enriched As described in Example 14, flask propagations were performed to estimate the potential inhibitory effect of ammonium molybdenate and the extent of molybdenum accumulation. See Table 29 for our data.

Table 29

Nutrient Mo content (mg / dm ³ ) OD recorded Mo / biomass (Pg / g) 0 10.55 20 12.80 2750 50 13.20 6900 100 15.05 14520 200 12.00 27,950 Ammonium molybdenate was grown in a fermenter at a concentration of 100 mg / dm ³ Mo in one installment, the exponential phase of propagation was added to the medium at the beginning, pH control was not performed. See Table 30 for our data.

Table 30 Time (H) OD Mo / fermentation broth (mg / dm ³ ) 0 1.50 2 1.95 - 3 2.40 98 4 3.30 76 6 4.25 69 8 4.50 64 12 7.85 55 24 13.00 12 Mo uptake: 3600 pg / g dried yeast

Feed yeast accumulates molybdenum as well as baker's yeast.

Example 17

Cobalt-enriched Saccharomyces cerevisiae A cobalt at various concentrations (10, 20, 50,

When cultured in media containing 100 and 200 mg / dm ³ ), the yeast was tested for inhibition of yeast growth by the cobalt content of the medium and the ability of the yeast to accumulate cobalt from media containing different concentrations of cobalt. Cobalt was added to the medium as cobalt (H) chloride. The results of flask propagation are summarized in Table 31.

Table 31

Media Co content (mg / dm ³ ) OD Recorded Co / biomass (pg / g) 0 2.85 - 10 2.20 250 20 1.90 310 50 1.75 820 100 1.70 3450 200 1.45 X

x: biomass was not recoverable

Because cobalt (II) chloride strongly inhibited the growth of yeast, only 20 mg / dm ³ Co was used in the fermentor. The salt was added to the broth every five hours from the start of the exponential phase of growth, with no pH control. See Table 32 for our measurement data.

Table 32

Time (H) OD Co / fermentation broth (mg / dm ³ ) 0 0.45 - 2 0.60 - 3 1.10 3.8 4 1.25 7.9 5 1.55 12.2 6 2.25 15.3 7 2.65 20.4 8 2.75 19.6 10 2.90 19.5 24 3.15 19.8

Co-uptake: 250 pg / g dried yeast enrichment: 80-fold

Example 18

Cobalt Enriched Candida utilis Flask propagations were performed as described in Example 17 to estimate the inhibitory effect of cobalt (H) chloride and the degree of cobalt accumulation. See Table 33 for our data.

Table 33

Nutrient Co-content OD Co / biomass absorbed (mg / dm ³ ) (pg / g)

0 11.45 10 11.90 235 20 11.05 590 50 8.15 1705 100 4.30 3410 200 2.65 11055

HU 205 379 Β

Since cobalt (II) chloride strongly inhibited the growth of yeast at concentrations of 50 mg / dm ³ and above, only 20 mg / dm ^{3 of} co-concentration was used in the fermentor. The salt was added in one portion at the start of the exponential phase of growth, no pH control. Our results are summarized in Table 34.

Table 34

Time (H) OD Co / fermentation broth (mg / dm ³ ) 0 1.90 __ 2 2.35 - 3 2.45 19.5 5 2.95 11.2 7 3.70 9.5 9 4.85 9.0 11 5.90 8.6 24 17.55 8.5

Co-uptake: 280 pg / g dried yeast

In the case of fermentation propagation, the co-uptake of both yeasts is approximately the same as that of the flask, but the reduction in atiter is not significant.

Example 19 30

Vanadium-enriched Saccharomyces cerevisiae

Vanadium in various concentrations (20, 50,

When grown in medium containing 100 and 200 mg / dm ³ ), the yeast was tested for inhibition of yeast growth by the vanadium content of the medium and the amount of vanadium that the yeast was able to accumulate in media containing different concentrations of vanadium. Vanadium was added to the medium as VOSO ₄ . The results of flask propagation are summarized in Table 35. 40

Table 35

Media V content (mg / dm ³ ) OD recorded V / biomass (Pg / g) 0 2.85 20 2.30 490 50 2.15 1150 100 1.70 1740 200 1.45 X

x: biomass was not recoverable 55

Fermentation propagation was performed at a concentration of 50 mg / dm ³ V. The VOSO ₄ salt was added in one portion to the rig at the start of the exponential phase of propagation, with no pH control. The results are shown in Table 36.

Table 36

Time (H) OD V / fermentation juice (mg / dm ³ ) 0 0.35 - 3 0.40 - 4 0.85 54 5 1.45 54 7 2.05 52 9 3.15 51 11 3.65 49 24 4.20 47

V-images 1460 pg / g dried yeast enrichment: 30,000-fold

Example 20

A19. except that during laboratory fermentation, VOSCL salt was added to the medium in five portions during the exponential phase of growth. Our results are a

Table 37 summarizes.

Table 37

Time (H) OD V / above (mg / dm ³ ) 0 0.45 - 2 0.60 - 3 0.85 9 4 1.15 20 5 1.70 35 6 1.95 42 7 2.35 50 9 3.10 48 11 3.60 46 24 4.65 44

V-uptake: 1520 pg / g dried yeast enrichment: 30,000-fold

In both examples, the vanadium uptake of baker's yeast resulted in an extremely high vanadium concentration compared to the vanadium content of normal baker's yeast.

Example 21

Candida utilis enriched with vanadium

Flask propagations were performed as described in Example 19 to estimate the inhibitory effect of VOSO ₄ and the degree of vanadium accumulation. Our data a

See Table 38.

HU 205 379 Β

Table 38

Media V content (mg / dm ³ ) OD Intake V / Biomass (pg / g) 0 9.35 - 20 8.75 920 50 7.50 1350 100 5.95 1810 200 4.60 2950

For enzyme propagation, a concentration of 50 mg / dm ³ V was used as in Example 19. VOSO _{4 was} added in one portion to the medium at the start of the exponential phase of propagation, with no pH control. Our results are shown in Table 39.

Table 39

Time (H) OD V / fermentation juice (mg / dm ³ ) 0 1.90 - 2 2.65 - 3 3.05 49 5 4.85 41 7 10.20 36 9 14.55 27 11 18.50 22 24 24.80 15

V-uptake: 1290 pg / g dried yeast

Like baker's yeast, Candida utilis accumulates vanadium very well.

Example 22

In the case of lithium-enriched Saccharomyces cerevisiae flask growth, the potential inhibitory effect of the salt used, in this case Li ₂ SO _4, on the growth of yeast was determined. The concentrations used were 20, 50, 100 and 200 mg / dm ³ Li, respectively. Table 40 shows only the optical density (OD) values reflecting the growth of yeast, since the uptake was below the limit of detection with the exception of 200 mg / dm ³ .

Table 40

Media Li content (mg / dm ³ ) OD Liquid uptake / biomass (pg / g) 0 2.85 20 2.85 - 50 2.70 - 100 2.25 - 200 1.80 <5

For fermentation propagation, Li ₂ SO ₄ corresponding to a concentration of 50 mg / dm ³ Li is added to the culture medium at the beginning of the exponential growth phase. No pH adjustment was made during propagation. See Table 41 for our measurement data.

Table 41

Time (H) OD Li / fermentation juice (mg / dm ³ ) 0 0.30 3 0.45 - 4 0.60 48 6 0.95 45 8 2.35 44 10 3.05 43 12 3.80 43 24 4.50 * 45

Li uptake: 8 pg / g dried yeast enrichment: 800-fold *: increase in concentration explained by cell autolysis

The lithium uptake of the yeast in the fermentor remains below 10 pg / g, but this value represents an increase of several hundred times that of the normal content of the normal yeast Li.

Example 23

Lithium-enriched Candida utilis

As described in Example 22, flask propagations were performed to inhibit or inhibit Li ₂ SO ₄ inhibition. to estimate the degree of lithium accumulation. See Table 42 for our data.

Table 42

Media Li content (mg / dm ³ ) OD recorded Li / biomass (Pg / g) 0 10.15 20 11.20 34 50 11.45 75 100 13.60 96 200 11.40 93

The growth of feed yeast was not inhibited by Li ₂ SO ₄ , so for fermentation growth we used Li concentration of 200 mg / dm ³ (Table 43). The salt was added in one portion at the beginning of the exponential phase of growth, with no pH control.

HU 205379 Β

43.táblázat

Time (H) OD Li / fermentation juice (mg / dm ³ ) 0 1.80 - 2 2.45 - 3 2.65 205 5 3.75 200 7 8.60 200 9 12.40 198 12 14.45 200 24 17.90 196

Li uptake: 70 yg / g dried yeast

In contrast to the baker's yeast, the growth of feed yeast was not inhibited by L2SO4, and the uptake of Li was also higher.

Example 24

Arsenic-enriched Saccharomyces cerevisíae

In the case of flask growth, the possible inhibitory effect of the salt used, in this case sodium arsenite, on the growth of yeast was determined. The concentrations used were 10, 20, 50 and 100 mg / dm ³ As, respectively. Table 44 shows the arsenic content of the yeast samples in addition to the optical density (OD) values.

Table 44

Media As content (mg / dm ³ ) OD recorded As / biomass (Gg / g) 0 2.85 - 10 2.90 95 20 2.55 175 50 2.20 330 100 2.05 885

Due to the inhibitory effect of arsenite, 50 mg / dm ³ As concentration was chosen for fermentation propagation. Sodium arsenite was added to the medium at the appropriate concentration at the start of the exponential phase of growth (Table 45). ApH was not regulated. Table 45 Time (H) OD As / fermentation juice (mg / dm ³ ) 0 0.45 2 0.60 - 3 0.95 54 5 2.35 54 7 3.65 52 9 4.50 51

Time (H) OD As / fermentation juice (mg / dm ³ ) 11 4.85 51 24 5.25 48

As-uptake: 230 yg / g dried yeast enrichment: 200-fold

Example 25

Arsenic Enriched Saccharomyces cerevisíae The procedure described in Example 24 was followed, except that sodium arsenate was used as the source of As instead of sodium arsenite.

Table 46

Media As content (mg / dm ³ ) OD Utilized As / Biomass (gg / g) 0 2.85 - 10 2.95 215 20 2.20 405 50 2.55 940 100 2.30 1720

Due to the inhibitory effect of arsenate, 50 mg / dm ³ As concentration was chosen for fermentation growth. Sodium arsenate was added at appropriate concentrations at the start of the exponential phase of growth (Table 47). ApH was not regulated.

Table 47

Time (H) OD As / fermentation juice (mg / dm ³ ) 0 0.40 - 2 0.75 - 3 0.95 50 5 1.35 51 7 2.70 51 9 3.55 49 11 4.05 48 24 4.60 47 As-up 4.80 gg / g dried yeast enrichment: 400-fold

A24. and 25, As inhibited slightly the growth of yeast but uptake of As was satisfactory. Sodium arsenate had a better accumulation than sodium arsenite.

Example 26

Silicon-enriched Saccharmyces cerevisíae

In the case of flask propagation we have found the occasion14

EN 205 379 salt, in this case sodium silicate, to inhibit the growth of yeast. The concentrations used were 40,200, 400 and 800 mg / dm ^{* 2 3} Si, respectively. Table 48 also shows the silicon content of the yeast samples in addition to the optical density (OD) values.

Table 48

Media Si content (mg / dm ³ ) OD Utilized Si / Biomass (pg / g) 0 3.15 - 40 3.30 710 200 3.45 795 400 3.50 1250 800 3.80 1620

The fermentation was first carried out at 50 mg / dm ³ Si concentration. The appropriate amount of sodium silicate was added in one portion at the beginning of the exponential phase of growth and no pH control was performed. See Table 49 for our data. Table 49 Time OD Si / broth (H) (mg / dm ³ ) 0 0.35 - 2 0.40 - 3 0.60 52 4 0.80 51 6 1.40 48 8 2.85 45 10 3.10 44 12 3.90 42 24 4.60 38

Si uptake: 1160 gg / g dried yeast enrichment: 40 fold

Example 27

Silicon-enriched Saccharomyces cerevisiae Fermentor growth by Si supplementation as described in Example 26, except that 100 mg / dm ³ Si of sodium silicate was added to the medium. The measurement results are summarized in Table 50.

Table 50

Time OD Si / fermentation broth (h) (mg / dm ³ )

0.30

0.40

0.55 103

0.90 10

Time (H) OD Si / fermentation juice (mg / dm ³ ) 7 1.25 98 9 2.60 95 11 3.50 92 24 4.65 89

Si uptake: 3200 gg / g dried yeast enrichment: 100 fold

Both examples show that, when grown in a well-aerated fermentor, yeast absorbs more silicon than shake flask growth. The Si uptake in the former case is almost proportional to the amount administered.

Example 28

Titanium enriched Saccharomyces cerevisiae

During growth in flasks, we examined the potential growth inhibitory effect of Ti-source titanium ascorbate chelate and the ability of yeast to accumulate titanium in its cells. The titanium ascorbate was added in an amount such that the Ti concentrations were 10, 50, 100 and 200 mg / dm ^{3, respectively} . See Table 51 for our results.

Table 51

Media Ti content (mg / dm ³ ) OD Ti / biomass uptake (pg / g) 0 3.45 10 3.30 540 50 3.50 8560 100 3.40 15,370 200 3.05 25,310

20 mg / dm ^{3 of} Ti-Total was added to the Feimentor within 2 hours of the onset of the exponential growth phase. Titanium ascorbate was immediately assimilated by yeast, as can be seen from Table 52. During propagation, the pH was set at 4 We maintained. Table 52 Time OD Ti / broth (H) (mg / dm ³ ) 0 0.30 2 0.45 - 3 0.70 2.4 4 1.15 1.8 5 1.60 4.5 6 2.30. 3.7 7 2.75 3.5

HU 205 379 Β

Time (H) OD Ti / fermentation broth (mg / dm ³ ) 9 3.90 3.5 11 4.35 3.2 24 4.85 3.5

Ti uptake: 1500 gg / g dried yeast enrichment: approx. 1000x

Example 29

Tellurium-enriched Saccharomyces cerevisiae Flask growth revealed the potential inhibitory effect of the salt used, in this case potassium telluride, on the growth of yeast. The concentrations used were 5,10,20 and 50 mg / dm ³ Te, respectively. Table 53 also shows the tellurium content of the yeast samples in addition to the optical density (OD) values.

Table 53

Nutrient solution Te content (mg / dm ³ ) OD recorded You / biomass (Gg / g) 0 3.10 - 5 2.85 1050 10 3.00 1750 20 2.95 3170 50 2.35 3240

For enzyme growth, a Te concentration of 20 mg / dm ³ was set at the start of the exponential phase of growth. The pH was not controlled. Our measurement data are summarized in Table 54.

Table 54 Time (H) OD You / fermene (mg / dm ³ ) 0 0.35 2 0.50 - 3 0.95 21 5 1.75 20 7 3.10 14 9 3.45 15 11 3.65 14 24 3.95 14

Te-uptake 2150 gg / g dried yeast enrichment: 2000 times

The data indicate that Te, when added to culture medium in the form of potassium telluride, slightly inhibits the growth of baker's yeast, but is well absorbed by the cells.

Example 30

When nickel-enriched Saccharomyces cerevisiae Flask growth was used, the potential inhibitory effect of nickel sulfate nickel sulfate on yeast growth was determined. The concentrations used were 10, 20, 50, 100 and 200 mg / dm ³ Ni, respectively. Table 55 shows the nickel content of the yeast samples in addition to the optical density (OD) values.

Table 55

Media Ni content (mg / dm ³ ) OD recorded Ni / biomass (Gg / g) 0 2.95 10 3.30 375 20 2.90 760 50 2.45 910 100 1.85 3750 200 1.20 X

x: there was no biomass available

Due to the high degree of growth inhibition, a nickel sulfate corresponding to a concentration of 20 mg / dm ³ Nl was added to the fermentor. The dosing was done in one increment at the start of the exponential phase of growth with no pH control (Table 56). Table 56 Time OD Ni / broth (H) (mg / dm ³ ) 0 0.45 - 2 0.55 - 3 0.70 18 5 1.10 16 7 1.95 15 9 3.10 11 11 3.80 10 24 5.30 11

Ni uptake: 290 gg / g dried yeast enrichment: 250 times

Example 31

In cesium-enriched Saccharomyces cerevisiae Flask growth, a possible inhibitory effect of Cs-source cesium chloride on the growth of yeast was determined. The concentrations used were 10, 20, 50, 100 and 200 mg / dm ³ Cs, respectively. Table 57 shows the cesium content of the yeast samples in addition to the optical density (OD) values.

HU 205 379 Β

Table 57

Media Cs content (mg / dm ^{* 2 3 4} ) OD Cs / biomass uptake (pg / g) 0 2.95 - 10 2.80 42 20 2.85 65 50 2.80 163 100 2.80 225 200 2.75 230

In yeast fermentation, baker's yeast was grown at a concentration of 100 mg / dm ³ Cs. The addition was made at the beginning of the exponential phase of growth and the pH was not adjusted. The results are shown in Table 58

together. Table 58 Time (H) OD Cs / fermentation juice (mg / dm ³ ) 0 0.35 2 0.40 - 3 0.55 100 5 1.15 100 7 2.45, 98 9 3.25 100 11 3.95 95 24 5.30 95

Cs uptake: 120 pg / g dried yeast enrichment: 100 fold

In the case of fermentation propagation, only half of the accumulation in the flask was achieved, but the degree of cesium enrichment was the same.

Example 32

Rubidium enriched Saccharomyces cerevisiae

Rubidium was not pre-tested in a flask because its physical and chemical properties and biochemical behavior are similar to those of cesium.

For enzyme propagation, the procedure described in Example 31 was followed except that rubidium chloride was used as the Rb source and the amount of Rb introduced was 200 mg / dm ³ . See Table 59 for our data.

Table 59

Time OD Rb / fermentation broth (h) (mg / dm ³ )

0.40

0.45

0.55,202

0.90.204

6 1.35 201 8 2.75 198 10 3.40 198 12 4.50 196 24 5.70 195

Rb uptake: 300 pg / g dried yeast enrichment: 300 fold

Rubidium enrichment developed as expected, baker yeast picking up metal in the order of hundreds, similar to cesium. The actual titer at higher dosing concentrations is higher than that of cesium.

Example 33

Iodine-enriched Saccharomyces cerevisiae

In the case of flask propagation, the possible inhibitory effect of potassium iodide as a source of iodine on the growth of yeast was determined. The concentrations used were 50, 100, 200 and 500 mg / dm ^3, respectively. Since potassium iodide at the above concentrations did not influence the growth of yeast and the recovered biomass was not sufficient to determine the iodine content, only the fermentor results are tabulated.

The yeast was grown in a fermenter at a dose of 200 mg / dm ³ , and was added in one increment at the start of the exponential phase of growth. There was no pH control in the process. Table 60 shows our data.

Table 60

Time (H) OD I / ml of fermentation broth (mg / dm ³ ) 0 0.35 2 0.45 - 3 0.70 205 5 2.30 205 7 3.95 203 9 4.60 200 11 5.15 198 24 5.65 196

I-uptake: 80 pg / g dried yeast enrichment: approx. 60 times

Example 34

Fluoride-enriched Saccharomyces cerevisiae Flask growth revealed the potential inhibitory effect of F-source sodium fluoride on yeast growth. The concentrations used were 5, 10, 20 and 50 mg / dm ³ F, respectively. Only optical density (OD) values are shown in Table 61, since fluorine content could not be measured from the low biomass.

HU 205 379 Β

Table 61

Media F content (mg / dm ³ ) OD Intake F / biomass (gg / g) 0 3.25 - 5 2.70 X 10 2.05 X 20 1.45 X 50 0.90 X

x: no. was able to extract biomass

Baker yeast was grown in fermenter at 20 mg / dm ³ F concentration. Dosing was performed in four increments every hour from the start of the exponential phase of growth, and the pH was not adjusted. Our results are a

Table 62 below.

Table 62

Time (H) OD F / fermentation juice (mg / dm ³ ) 0 0.40 2 0.55 - 3 1.05 7 4 1.35 11 5 1.45 15 6 1.55 18 7 1.70 15 9 1.85 14 11 2.15 14 24 2.60 12

F-uptake: 96 gg / g dried yeast enrichment: 90-fold

Example 35

Fluorine Enriched Saccharomyces cerevisiae The procedure described in Example 34, except that ammonium fluoride was added to the culture medium as a source of fluorine instead of sodium fluoride. Measurement data for flask propagation are shown in Table 63.

Table 63

Media F content (mg / dm ³ ) OD Intake F / biomass (gg / g) 0 3.75 10 3.45 17 20 2.90 40 50 1.75 X

x: there was no biomass available

Also for enzyme propagation, the procedure described in Example 34 was followed. Ammonium fluoride was added in four portions to the medium so that the total fluorine concentration was 20 mg / dm ³ . The measurement results are shown in Table 64.

Table 64

Time (H) OD F / fermentation juice (mg / dm ³ ) 0 0.50 2 0.65 - 3 1.45 4 4 2.00 7 5 2.60 11 6 2.75 15 7 3.10 14 9 3.55 15 11 3.80 14 24 3.65 14

F-uptake: 58 gg / g dried yeast enrichment: 50-fold

In contrast to iodine, fluorine strongly inhibited the growth of baker's yeast.

Claims (1)

PATIENT PERSONALITY Process for the production of micronutrients, namely iron, copper, zinc, manganese, molybdenum, cobalt, vanadium, lithium, arsenic, silicon, titanium, tellurium, nickel, cesium, rubidium, iodine or fluorine enriched yeast, in particular Saccharomyces cerevisiae or Candida utilis. to determine which salt of the particular micro-nutrient is to be used, the nature and amount of the salt selected depending on the effect of the particular salt of the microelement on reproduction; then the yeast cells are propagated in a known medium in a manner known per se, while the selected water soluble salt of the microelement together with the nutrient solution or during the propagation at the beginning of the exponential phase or in the exponential phase, simultaneously or in several portions or continuously, predetermined the amount of the administered portion, the mode of administration and time. ; After propagation, the cell mass is separated, washed with water, filtered and, if desired, dried, within which a) In the case of iron-enriched yeast, iron (II) sulphate or iron (fll) citrate or iron (1H) -ammonium citrate is used in an amount of 50-100 mg Fe / dm ³ medium and is added at the beginning of the propagation at any of the yeasts. ; b) For copper-enriched yeast, copper (H) sulfate is used at 3-15 mg Cu / dm? stock solution EN 205 379 Β S. cerevisiae and is administered simultaneously at the beginning of the exponential phase or in multiple portions in the exponential phase and at a dose of 50-100 mg of Cu / dm ³ medium at the start of the exponential phase at Candida utilis; c) for zinc-enriched yeast, zinc sulfate is used in an amount of 10-250 mg of Zn / dm ³ medium and is administered simultaneously at the beginning of the exponential phase at S. cerevisiae and C. utilis; d) in the case of manganese-enriched yeast, manganese sulphate is used in an amount of 50-150 mg Μη / dm ³ and is added in 3 to 5 increments in the exponential phase at S. cerevisiae and 50-300 mg Μη / dm ³ at the same time, exponentially administered. at the beginning of the phase at Candida utilis; e) in the case of molybdenum-enriched yeast, ammonium molybdenum is used in an amount of 10-300 mg Mo / dm ³ medium and is administered simultaneously at the beginning of the exponential phase at S. cerevisiae and C. utilis; f) in the case of cobalt-enriched yeast, cobalt (II) chloride is used in an amount of 10 to 100 mg of Co / dm ³ medium and is administered in 3-5 portions in the exponential phase at S. cerevisiae and administered simultaneously at the beginning of the exponential phase at Candida utilis; g) for vanadium-enriched yeast, vanadyl sulfate is used in an amount of 20-100 mg of V / dm ³ medium and is administered simultaneously at the start of the exponential phase or in 3-5 portions in the exponential phase at S. cerevisiae and C. utilis; h) for lithium-enriched yeast, lithium sulfate was used and co-administered at the beginning of the exponential phase in an amount of 50-100 mg Li / dm ³ medium at S. cerevisiae and 50-300 mg Li / dm ³ medium at Candida utilis; i) using arsenic-enriched yeast sodium arsenite or sodium arsenate and adding 50-100 mg of As / dm ³ medium in one portion at the beginning of the exponential phase at S. cerevisiae and C. utilis; j) for silicon-enriched yeast, sodium silicate is used in an amount of 20-500 µl Si / dm ³ medium and is added in one portion at the beginning of the exponential phase at S. cerevisiae and C. utilis; k) for titanium-enriched yeast, titanium ascorbate is used in an amount of 10-100 mg Ti / dm ³ and added in one portion to the exponential phase at S. cerevisiae and C. utiEs; (l) in the case of the yeast-enriched yeast, potassium tellurite is used in an amount of 5-50 mgTe / dm ^{3 of the} medium and is administered simultaneously at the beginning of the exponential phase at S. cerevisiae and C. utilis; m) in the case of nickel-enriched yeast, nickel sulfate is used in an amount of 10-100 mg Ni / dm ³ medium and is administered simultaneously at the beginning of the exponential phase at S. cerevisiae and C. utilis; n) in the case of cesium-enriched yeast, cesium chloride is used in an amount of 50-200 mg of Cs / dm ³ medium and is administered simultaneously at the beginning of the exponential phase at S. cerevisiae and C. utilis; o) in the case of yeast enriched with rubidium, rubidium chloride is used in an amount of 50-200 mg of Rb / dm ³ medium and is simultaneously administered at the beginning of the exponential phase at S. cerevisiae and C. utilis; p) for iodine-enriched yeast, potassium iodide is used in an amount of 50-300 mg of I / dm ³ medium and is administered simultaneously at the beginning of the exponential phase at S. cerevisiae and C. utilis; r) in the case of fluorinated enriched yeast, sodium fluoride or ammonium fluoride is used in an amount of 5-20 mg of F / dm ³ medium and is added in 3-5 parts in the exponential phase at S. cerevisiae and C. utilis.