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Patent 2649145 Summary

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(12) Patent: (11) CA 2649145
(54) English Title: A METHOD OF PRODUCING SELENIUM-ENRICHED YEAST PRODUCTS, AND USES THEREOF
(54) French Title: UNE METHODE DE PRODUCTION DE PRODUITS DE LEVURE ENRICHIS AU SELENIUM ET LEURS UTILISATIONS
Status: Granted
Bibliographic Data
(51) International Patent Classification (IPC):
  • C12N 1/16 (2006.01)
  • A23L 33/14 (2016.01)
  • A23L 33/16 (2016.01)
  • A61K 33/04 (2006.01)
  • C12N 1/18 (2006.01)
  • A21D 8/04 (2006.01)
(72) Inventors :
  • MIROSHNYCHENKO, OLEKSANDR (Canada)
  • MIROSHNYCHENKO, ZORYNA (Canada)
(73) Owners :
  • MIROSHNYCHENKO, OLEKSANDR (Canada)
(71) Applicants :
  • MIROSHNYCHENKO, OLEKSANDR (Canada)
(74) Agent:
(74) Associate agent:
(45) Issued: 2013-09-24
(22) Filed Date: 2008-12-22
(41) Open to Public Inspection: 2010-06-22
Examination requested: 2009-02-17
Availability of licence: N/A
(25) Language of filing: English

Patent Cooperation Treaty (PCT): No

(30) Application Priority Data: None

Abstracts

English Abstract


The present invention describes a process for preparing selenium-enriched
yeast products
that have high contents of organically bound selenium. It is achieved by
adding the selenium
compound to the nutrient medium wherein formation of multiple enzyme-metal
complexes is
optimized, which increases the maximum rate of the fermentation process and
boosts the
concentration of organic selenium in these products. The invention also
provides a means for the
production of practically non-toxic selenium-enriched yeast products for use
in foods, dietary
supplements, or drugs.


French Abstract

La présente invention décrit une méthode de production de produits de levure enrichis au sélénium qui ont une teneur élevée en sélénium organique. La méthode est réalisée en ajoutant le composé de sélénium au support de nutriment où la formation de multiples complexes enzyme-métal est optimisée, ce qui diminue le taux maximal du processus de fermentation et augmente la concentration de sélénium organique dans ces produits. L'invention fournit également une méthode de production de produits de levure enrichis au sélénium pratiquement non toxiques pouvant servir dans les aliments, les suppléments alimentaires ou les médicaments.

Claims

Note: Claims are shown in the official language in which they were submitted.


16
What is claimed is:
1. A method for producing a selenium-enriched yeast products having an
organically bound
selenium content of between approximately 3000 ppm and 5000 ppm, calculated on
a dry
matter in the yeast, comprising the steps of:
(a) preparing trace element composition for a 5 L nutrient medium as NaCl
(0.2045 g),
KH2PO4 (0.2755 g), CaCl2 .cndot 2H2O (0.2054 g), NH4Cl (2.1094 g), MgSO4
.cndot 7H2O
(1.3662 g), FeSO4 .cndot 7H2O (0.567 mg), Na2B407 .cndot 10H2O (18.55 µg),
ZnSO4 (1.074
mg), CuSO4 .cndot 5H2O (0.375 mg), Co(NO3)2 .cndot 6H2O (29.10 µg),
(NH4)6Mo7O24 .cndot 4H2O
(0.2649 mg), NiSO4 .cndot 6H2O (10.52 mg), MnSO4 .cndot H2O (1.014 mg), KI
(0.1667 mg),
biotin (0.60 mg);
(b) preparing an aqueous yeast grown fermentable sugar containing nutrient
medium in
which molasses is the sole carbon source;
(c) adjusting the pH of the nutrient medium to 4.9-5.0;
(d) adjusting the temperature of the aqueous nutrient medium to 37-38
°C;
(e) adjusting the aeration rate to the nutrient medium;
(f) adding yeast to the nutrient medium;
(g) attaining a pH of about 4.45, at which formation of multiple enzyme¨metal
complexes are optimized;
(h) preparing an aqueous solution of selenite salt;
(i) adding the selenite solution in the range of 0.13 to 0.20 g/l to the
nutrient medium at a
pH about 4.45;
(j) cultivating for a period from about eight (8) to about (18) hours while
gradually
decreasing the pH to about 4.0;
(k) adjusting the pH with ammonium hydroxide to 4.6-4.7 when the pH of the
fermentation reaction reaches 4.0 during cultivation;
(l) harvesting the selenium yeast product and concentrating it by
centrifugation in order
to separate the selenium yeast product from the nutrient medium; and
(m) washing the selenium yeast product.
2. The method of claim 1, further comprising a step of drying the washed the
selenium yeast
products for use as large-scale baker's or other manufactured yeasts.

17
3. The method of claim 1, further comprising a step of pasteurizing and drying
the washed
the selenium yeast products for use in dietary supplements and drugs.
4. The method of claim 1, wherein said harvested selenium-enriched yeast
products are
washed until achieve a negative reaction with SnCl2, which indicates that
water-soluble
residual selenium compounds are removed.
5. The method of claim 1, wherein said selenite salt is Na2Se2O3.
6. The method of claim 1, wherein said the yeast growth on carbon source
comprises one of
the members of the group selected from beet molasses or cane molasses.
7. The method of claim 1, wherein said the yeast growth carbon source
comprises a mixture
of beet and cane molasses.
8. The method of claim 1, wherein said yeast selected is baker's yeast
(Saccharomyces
cerevisiae).
9. The method of claim 1, wherein said yeast selected is brewer's yeast
(Saccharomyces
uvarum).
10. The method of claim 1, wherein said the airflow rate on oxygen transfer
for a 5 L nutrient
medium is 9 L/min and agitation rate is 250 rpm.

Description

Note: Descriptions are shown in the official language in which they were submitted.


CA 02649145 2012-12-18
1
A METHOD OF PRODUCING SELENIUM¨ENRICHED YEAST PRODUCTS,
AND USES THEREOF
FIELD OF THE INVENTION
The present invention relates to a method of producing selenium-enriched yeast
products
having high content of organically bound selenium for use in foods, dietary
supplements, or
drugs.
BACKGROUND OF THE INVENTION
Selenium is a very important essential trace element for humans, and its
deficiency or
excess has been implicated in the development of several diseases. It is
incorporated into
proteins, and presently about 35 selenoproteins have been identified, though
many have roles
that have not yet been fully elucidated [Behne D., 2000]. The known biological
functions of
selenoproteins include, for example, the protection of cell membranes against
oxidative damage
(glutathione peroxidase) [Rotruck J.T., 1973)] and the regulation of thyroid
hormone metabolism
[Arthur J.R., 1993; Corvilain B., 1993]. Selenium deficiency is believed to be
the cause of
Keshan disease in northeastern and southwestern China and in the Transbaikalia
region of
southern Siberia [Reilly C., 1996; Keshan Disease., 1979]. Selenium has been
known to prevent
coronary heart disease [Neve J., 1996: Salonen J.T., 1982] and has a very
specific role in
spermatogenesis that is essential for male fertility [Oldereid N.B., 1998;
Rayman M., 2000;
Pfeifer H., 2001]. Selenium has additional important health benefits,
particularly in relation to
the immune response [Kiremidjian-Schumacher L., 1994; Taylor E.W., 1999] and
cancer
prevention: prostate, lung, colon, and possibly, breast cancer [Clark L.C.,
1996; Yoshizawa K.,
1998; Knekt P., 1998; Decensi A., 2000; Combs Jr G.F., 2001; Lopez-Saez J.B.,
2003; Rejali L.,
2007; Peters U., 2007]. Selenium deficiency has also been observed in patients
with AIDS and
has predictive value with respect to both the rate of development and
resulting mortality from
this disease [Patrick L., 1999; Baum M.K., 1997]. However, when in relatively
low
concentrations, selenium provides beneficial health effects; at higher
concentrations selenium

CA 02649145 2012-12-18
2
exhibits dramatic toxicity. Symptoms in people with high dietary intakes of
selenium include
gastrointestinal disturbances, nausea, vomiting, discoloration of the skin,
tremor, numbness in
limbs, tooth decay and a garlic breath odor [Hogberg J., 1986; Yang G.Q.,
19831. It was
hypothesized that selenium intake in the range of 40-100 tig/d primarily has
antioxidant and
immune-strengthening effects, while intake in the range 200-500 ggid has
specific cancer-
preventive properties [Rayman M.P., 2002].
Foodstuffs constitute the main source of selenium for people and animals
through the
food chain. However, intakes of this magnitude may be problematic due to the
low concentration
of selenium in common foods [Morris V.C., 1970]. As a result, additional
source of safe and
efficient supplements of selenium should be used. Although most water-soluble
inorganic
selenium compounds and selenium organic compounds from food are effectively
absorbed in the
gastrointestinal tract [Bopp B.A., 1982], it is important to realize that
bioavailability is not the
same as bioaccessibility (amount of the ingested element that becomes
avaliable for absorption
in the gastrointestinal tract). Uptake and retention of inorganic selenium
salts are less effective
than that of organic selenium [Thomson C.D., 1986; Swanson C.A., 1991]. Using
elemental
selenium (Se(0)) particles additionally requires a carrier molecule that has
the capability of
forming a conjugate with Se(0) particles in the composition [Sieber et al., US
7,205,002 B2].
Ideally, selenium should be supplemented in the form (or forms) in which it
occurs in major
staple foods. More than 60 % of total selenium in corn, wheat and soybeans
consists of L(+)-
selenomethionine; and a y-glutamyl-Se-methylselenocysteine is a major organic
selenium
compound in Alhum plants [Whanger P.D., 2002]. Despite the fact that Se-
enriched garlic
reduced total tumor growths better then Se-yeast, supplementation of Se-garlic
in the diet at
different levels consistently caused a lower total tissue selenium
accumulation when compared to
Se-yeast [Ip C., 2000]. The selenomethionine is the most appropriate
supplemental form of
selenium because this aminoacid can be stored in the body, being incorporated
in general
proteins. It is a stable and safe storage mode for Se and it can later be
liberated from the proteins
as various Se-species [Suzuki T., 2005]. Additionally, the selenomethionine is
extensively
recirculated and passed through the liver, pancreas and peripheral tissues
many times before
being excreted [Patterson B.H., 1993; Veillon C., 19901. The selenomethionine
also detected in
human milk [Michalke B., 1997].

CA 02649145 2012-12-18
3
Organic forms (e.g. high-selenium yeast) are often preferred as supplements,
partly
because they are less acutely toxic and have higher bioavailability and
bioaccessibility than
inorganic supplements [Baerwald G., 1994; Malbe M., 1995]. Baker's yeast is
often chosen
because selenium compounds could be produced in significant quantity under
controlled
conditions and the product is known to contain a highly bioactive organic form
of selenium -
L(+)-selenomethionine - that is mainly incorporated in the proteins, and
constitutes about 60% of
total selenium content in the yeast [Kotrebai M., 1999; Larsen E.H., 2003].
Additionally, the
other important form among selenium organic compounds ¨ y-glutamyl-Se-
methylselenocysteine
was found in selenium yeast, and is considered to be an anticancer compound
[Infante H.G.,
2005].
Selenomethionine is being produced analogously to methionine as was evident
from the
fact, when a mutant strain of yeast that was unable to synthesize methionine,
had failed to
produce selenomethionine when grown in selenium containing media [Mason A.C.,
1994]. A
sulfate transporter [Cherest H., 1977] mediates sulfur uptake in yeast, so we
hypothesize that
assimilation of selenium in yeast should occur in a similar way.
Major clinical trials proved that dietary supplementation with
selenium¨enriched yeast
decreased cancer incidence and mortality rates by almost 50 % [Clark
L.C.,1996]. Currently
available commercial products for medical use typically contain up to 3,000
lig Se g4 [Demirci
A., 1999].
A number of patents describing the cultivation of selenium yeast contain
practical
limitations, which include low selenium content, relatively high extracellular
concentration of
inorganic selenium, and high cost of the final product. Ideally, high
extracellular concentrations
of selenium have to be avoided, whereas higher intracellular concentration of
selenium in the
organic form is preferred for administration to humans. For example,
Nagodowithana et al. [US
4,530,846] describes a method for producing a selenium-enriched yeast that
yield product with a
moderate intracellular selenium content of about 1,000 ppm; however, the
authors of this patent
say that "While intracellular selenium contents of yeasts are preferably in
the range of 1,000
ppm, or more, even as high as 2,500 ppm, the process has, as its practical
limitations, the
capacity of the yeast to assimilate the selenium during the yeast growth cycle
without adverse
effects on yield due to the selenium additive to the nutrients".

CA 02649145 2012-12-18
4
Moesgaard S. et al. [US 2005/0089530 Al] teach that for "preparing a yeast
product
containing significant and homogeneous amount of digestible, organically bound
selenium
= ...glucose and/or maltose being the only sources of carbon in the feeding
medium". Glucose
and/or maltose are expensive and thus have negative impact on the final cost
of the product.
Cheung [US 72049881 has cultivated yeast cells in an electromagnetic field to
produce the final
product, which led to substantial increase of its price as well.
Based on cited prior art we concluded that it is still essential to develop a
method of
preparation selenium-yeast product that provides:
(1) a high growth rate of selenium-enriched yeast using a cheaper source of
carbon in the
feeding medium;
(2) high concentration of organic binding selenium highly methabolizable by
the human
body, and thus suitable for dietary supplementation or therapy;
(3) low toxicity due to low extracellular concentration of inorganic selenium
in the
selenium-enriched yeast product;
(4) simplicity of the technological process of fermentation by using a one-
step continuous
fermentation method.
This patent addresses all the above mentioned points.
OBJECTS AND SUMMARY OF THE INVENTION
The selenium yeast can be prepared using a number of species. However,
Saccharomyces
cerevisiae is generally considered to be the most suitable for human
ingestion, and it is widely
used for preparation of bread and alcohol. The fermentation of the yeast by
controlling pH,
temperature, and aeration to optimal growth of the yeast strain and maximal
biomass production
has been well documented in the literature [Babeva I.P., 1979; Reed G., 1991;
Walker G.M.,
1998]. Numerous studies have been conducted to determine 'optimum'
concentration of the
various ionic species for growth [e.g. Jones R.P., 1984; Jones R.P., 1990;
Chandrasena G.,
1997]. The medium for yeast growth, which is various sugars to which vitamins,
nutritional salts
and Se salt, mostly as sodium selenite, as the Se source are added, has been
extensively reviewed
in the literature [e.g. Barnett J.A., 19761. The amount of Se in the cells
increases with increasing

CA 02649145 2012-12-18
the concentration of Se, however, the more Se is added initially, the stronger
the yeast growth is
inhibited [Ponce de Lecin C.A., 2002]. Usually, nutritional salts and Se salt
are added at several
= stages in the growth cycle [Nagodowithana et al., US 4,530,846; Fan et
al., US 6,368,643 B1].
The critical point of the fermentation reaction is the formation of the
enzyme's active site.
The import of additional metal ions during the fermentation process
[Nagodowithana et al., US
4,530,846; Moesgaard et al., US 2005/0089530 Al] will lead to attaching of
these ions,
especially transitional metal ions, to another part of the enzyme molecule and
the formation of
new complexes, thereby leading to change in the structure and shape of the
enzyme. The change
in shape means reducing the enzyme ability to bind with the substrate
correctly. This reduces the
concentration of 'active' enzyme resulting in a decrease in the maximum rate
of a chemical
reaction without changing the apparent binding affinity of the catalyst for
the substrate. Thus,
increasing the concentration of the substrate still does not allow the maximum
enzyme activity
rate to be reached.
In the present invention, increases of the rate of fermentation reaction and a
concentration
of organic binding selenium have been achieved by increasing uptake capacity
of the selenium
by the yeast. Inorganic selenium introduced after the formation of the
multiple enzyme¨metal
systems.
DESCRIPTION OF THE DRAWINGS
The present invention will be better understood and explained hereinafter with
reference
to the attached drawing, wherein:
FIG.1 represents kinetic curves of yeast cultivation. (- - -) yeast
cultivation without
selenium salt; ( ____ ) cultivation of selenium-enriched yeast product.
FIG.2 represents the dependence of the maximum rate of the fermentation
reaction on the
initial concentration of Na2Se03. (1) without selenium salt; (2) with a 0.13
g/1 concentration of
Na25e03; (3) with a 0.20 g/1 concentration of Na2Se03.

CA 02649145 2012-12-18
6
DETAILED DESCRIPTION OF THE INVENTION
Before the present invention is disclosed and described, it is to be
understood by all
skilled in art that this invention is not limited to the particular process
steps and materials
disclosed herein, because such process steps and materials may vary somewhat.
The broad aspect
of this invention is to assimilate various inorganic compounds by yeast in
order to produce not
only selenium-enriched yeast, but also to produce germanium-enriched yeast, or
with any
desirable cation or anion, other types of yeast, where step of adding the
selenium salt solution is
replaced by addition of the solution of germanium compounds, or any other
cation or anion of
inorganic salts to the fermenter after formation of the multiple enzymes¨metal
systems.
Yeasts require a number of inorganic ions in micro- and millimolar
concentrations for
optimum growth. Thus, Zn2+, Co2+, Mn2+, Mo5+, and Cu2+ are common catalytic
centers of an
enzyme [Jones R.P., 1984; Van Ho A., 2002]; Mg2+ or IC+ act as an activator or
stabilizer of
enzyme function [Wyatt H. V., 1964]; Ca2+ stabilizes yeast plasma membranes
[Lewis M.J.,
1978]. Phosphate as the species H2PO4- is recognized as critical for cell
growth [Soumalainen H.,
1971]. In addition, low concentrations of S042- [Schultz A.S., 1950], Cl- and
I- are also essential
for yeast growth [Morris E.O., 1958; Jones R.P., 1984].
Oxygen is an essential nutrient in aerobic fermentations. In contrast to other
nutrients, it
is the most difficult to supply because of its low solubility in water. One
gram of oxygen is
required for the formation of 1 gram of dry yeast mass [Mateles R.I., 1971].
However, increasing
concentration of oxygen leads to the formation of alcohol whose toxicity on
growth of a baker's
yeast was studied in Letourneau's research [Letourneau F., 1987]. Thus,
increasing concentration
of oxygen in the sparging gas during the culture of baker's yeast is limited
to 63%, at higher
concentration ethanol is formed [Oura E., 1974]. Based on this, the required
oxygen is usually
supplied to the fermenter by aeration additionally with an agitation for
better dispersion of the
bubbles of air and hence increasing surface area for oxygen transfer.
The fermentation must be glucose limiting because baker's yeast is sensitive
to glucose
concentration above 0.2% [Reed G., 1991]. In that case, concentration of
ethanol, as
intermediary product of metabolism, which formed during the fermentation, will
not affect
baker's yeast growth. In the present art, the beet molasses were chosen as
source of carbon
because only trace concentration of glucose is present in them [Steg A.,
1985]. We found that

CA 02649145 2012-12-18
7
use of other low glucose sugar sources, such as combination of cane molasses
with beet
molasses, to be satisfactory.
= Environmental parameters such as pH and temperature are also critical.
Baker's yeast
generally grows at controlled temperature. Growth rates, as expressed in
generation times were
determined to be 2.2 hr at 30 C, 2.1 hr at 36 C and 4 hr at 40 C [White J.,
1954]. However, this
value cannot be literally translated into practice because they reflect
generation times at the start
of the fermentation. The optimum pH level is between pH 4.5 to 5; however,
lower pH levels
increase the oxidation rate of the carbohydrates [Bhatnagar R.P., 1978].
The major steps in the fermentation of the selenium yeast include binding of
oxygen,
assimilation of ammonia and selenium, and oxidation. Metallo-enzymes catalyze
all of these
steps. Trace amounts of transition metals promote attaching substrate to the
enzyme's active site
with formation of the 'active¨substrate' complexes. Thus, the lower oxidation
states of Fe2+ and
Cu2+ have a strong tendency to bind oxygen. The oxidation reaction of the
carbohydrate with
variable-valence metal ions indicates first order of reaction with respect to
each reactant that
rises in acid solution [Krupenskii V.I., 1980, 1986; Bhatnagar R.P., 1978].
The formation of a
complex between D-xylose and Mn (III) is the determining step in the oxidation
of the
carbohydrates [Bhatnagar R.P., 1978]. Using spectrofotometric methods has
shown that
carbohydrates form complexes with cation oxidation agent preferably in
composition 1:1
[Krupenskii V.I., 1980]. Uptake of molibdate into yeast cell by sulfate
transporter [Fitzpatrick
K.L., 2008] is important for the formation of molibdenum-iron enzyme which is
the biosynthetic
pathway to assimilation of ammonia by yeast [Umbarger H.E., 1978].
However, transition metals yeast intake is dependent on concentration and
nature of these
metals. Metals form different types of chelates might not only increase the
rate of the oxidation
reactions but might also retard them. Toxic effects include blocking of
functional groups of
enzymes, inhibition of transport systems for essential ions and nutrients,
displacement and
substitution of essential metal ions from cellular locations, denaturation of
enzymes, and
disruption of membrane structure [Ochiai E.I., 1987]. For example, in the
oxidation reaction, the
function of essential Mn (II) [Jones R.P., 1984] has been found to be an
initiator of the formation
of hydroperoxide by reaction of R02. with Mn+2 ions, but the excess of free
Mn+2 efficiently
deactivates the intermediate complex [Zakharov I.V., 1998]. The salt Na2Mo04 =
H20 reduced
yeast growth at pH 5 in the first 8h [Crans D.C., 2004]. The presence of
copper ions is important

CA 02649145 2012-12-18
8
to binding oxygen and in the oxidation reaction, but its excess can cause cell
death during the
logarithmic growth phase [Jones R.P., 1984].
= The high catalytic activity of chelate compounds of transition metals was
found to depend
on both the ligand dentate number and on the three-dimensional structure of
the chelate core.
However, the latter is the major factor in the determination of the catalytic
activity of chelates
[Zakharov A.N., 1992; Solozhenko E.G., 1986]. The tetragonal distortion of a
complex of d-
transition metals influences the ability to combine with molecular oxygen
[Shevchenko Yu. N.,
1986]. Decreasing the number of coordinating atoms of the ligands with the
retention of the
coordination geometry of the chelate center leads to the enhancement of the
catalytic activity of
the Cu2+ chelate due to an increase in its stereochemical liability in a model
reaction of liquid-
phase oxidation by molecular oxygen [Zakharov A.N., 1994]. The molybdenum-
monoalkylhydroxylamine ligand inhibits yeast growth, while the molybdenum-
dialkylhydroxylamine ligands have little effect on yeast growth [Crans D.C.,
2004]. Thus, the
correlation should exist between the structure of the intermediate complex and
the kinetic
parameter of the product formation. As a result, activation of the parameters
that have the highest
influence on the formation of the enzyme's active site (catalytic activities
of chelates), such as
control of pH, temperature, and aeration, allows optimization of the formation
of multiple
enzyme¨metal systems and, as a result, leads to maximum high-selenium yeast
biomass
production.
In the present invention, the high catalytic activity of the enzyme (the
optimum of the rate
of forming of multiple enzyme-metal systems of fermentation reaction) was
achieved by
optimization of pH, the rate of the aeration, and addition of exact amount of
catalytic cations.
The kinetic measurements were carried out by controlling of pH during
fermentation. The kinetic
graph curves of fermentation are S-shaped in character (Fig. 1). The 3-phase
growth curve
responds to a formation of multiple enzyme-metal systems, necessary for growth
(a), a period of
growth at higher concentrations of multiple enzyme-metal systems (b), and then
allosteric
inhibition (c) [Wyatt H.V., 1964]. The induction period (a) appearing on the
kinetic curves with
the maximum fermentation rates after the inflection clearly demonstrates
accumulation of the
active particles during the initial period. The initial period sharply depends
on the rate of aeration
and agitation. The optimum rate for aeration in 5 L reactor was 9 L/min with
an agitation at 250
rpm. However, in large commercial fermenter rate of the aeration and an
agitation should be

CA 02649145 2012-12-18
9
optimized to achieve better dispersion of the air bubbles and hence increasing
surface area for
oxygen transfer. The optimal pH value during the cultivation should be
maintained between 3.9
= and 4.9, preferably between 4.0 and 4.7. The kinetic curves of the
fermentation of selenium-
enriched yeast also are S-shaped in character, as can be seen from Fig. 1, and
after the point of
inflection are satisfactorily linearized in the coordinates of a first order
equation with respect to
pH. The increase of the initial concentration of Na2Se03 decreases the maximum
rate of the
fermentation reaction (Fig. 2).
A typical method of cultivating the selenium yeast includes the following
steps:
(1) preparation of aqueous nutrition medium;
(2) preparation of aqueous solution of selenium salt;
(3) adjusting pH of the nutrition medium;
(4) adjusting rate of air supply;
(5) formation of the multiple enzyme¨metal systems;
(6) addition of the selenium solution to the feeding medium with active
multiple enzyme¨

metal systems;
(7) cultivation;
(8) harvesting;
(9) washing and pasteurization (if desired);
(10) drying of the yeast.
The nutrition medium is prepared by dissolving beet molasses in water,
addition of
vitamins (mostly biotin), and minerals including ammonia salt at the
temperatures between 36
and 37 C. Beet molasses usually contains most of vitamins in sufficient
quantity required for
baker's yeast growth, except biotin, which concentration is usually 0.01-0.02
ppm [Reed G.,
19911. As a result, biotin requires an external supply and should be added as
the synthetic
compound. As an alternative, the use of cane or citrus molasses to produce
beet/cane or
beet/citrus blends for yeast production could be considered. Additionally,
thiamin and vanadyl
salts can also be added in order to improve fermentation activity [Reed G.,
1991; Willsky G. R.,
1984]. After that, the pH was adjusted to 4.9 using HC1 and atmospheric air
supplied at 9 L/min
and agitated at 250 rpm. Because a maximum rate of fermentation reaction is
achieved after
formation of the multiple enzyme¨metal systems, the pH of the growth medium
was used as an
indicator, and therefore it was controlled and adjusted during the growth so
as to maintain the pH

CA 02649145 2012-12-18
between 4.0 and 4.7 by using ammonium hydroxide (Fig.1). After the growth
cycle is
completed, the yeast was harvested using a centrifugation to separate the
cultivated yeast from
= the nutrient medium and then washed to remove the excess of the
extracellular nutrients
including selenium salts in the yeast cream. The washing is carried out by
adding water followed
by centrifuging so that a yeast cream is produced. In the present process, the
yeast was washed
six times. The supernatant solution was removed and analyzed for the presence
of inorganic
selenium. The absence of inorganic Se in yeast preparations was indicated by
the knowledge that
both Se042- and Se032- can be reduced to elemental Se, a reddish precipitate,
by SnC12. Organic
Se does not form elemental Se under these conditions [Moesgaard S., 20011.
After washing, the
yeast was freeze-dried and total selenium analysis was determined by digesting
yeast in nitric
acid and finished using ICP-OES.
In the large-scale preparations of dietary supplements or drugs, before drying
product by
any conventional methods of drying organic materials (e.g. freeze-drying, drum
drying, tray
drying or spray drying), the yeast should be pasteurized at 85 C to kill the
yeast cells so that the
selenium present therein becomes available for the human digestion. In large-
scale preparations
of active dry selenium enriched yeast (e.g. baker manufacturing) tunnel drying
is preferable in
order to prevent losses in viability and fermentation activity [Belokon V.N.,
1962].
EXAMPLES
The following examples illustrate the results obtained by the use of the
method of the
present invention, but should not be constructed as limitations of the present
invention, and
should merely describe how to assimilate inorganic compounds by yeast after
formation of the
multiple enzyme¨metal systems based upon current experimental data.
The yeast growth nutrients were prepared as follows: 200 g beet molasses
containing 85
% of dry matter (50 % sucrose and 35 % "non-sugar") were diluted to 1 L with
distilled water,
then the nutrient medium was added. The nutrient medium had the following
initial composition:
NaC1 (0.2045 g), KH2PO4 (0.2755 g), CaCl2 = 2H20 (0.2054 g), NH4C1 (2.1094 g),
MgSO4 =
7H20 (1.3662 g), FeSO4 = 7H20 (0.567 mg), Na2B407 = 10H20 (18.55 ug), ZnSO4
(1.074 mg),
CuSO4 = 51120 (0.375 mg), Co(NO3)2 = 6H20 (29.10 pg), (NH4)6M07024 = 41420
(0.2649 mg),

CA 02649145 2012-12-18
11
NiSO4 = 6H20 (10.52 mg), MnSO4 = H20 (1.014 mg), KI (0.1667 mg), biotin (0.60
mg). Then the
final solution was diluted with distilled water to 5 L, and the pH adjusted to
4.9 using aqueous
= concentrated HC1. Temperature was maintained at about 37-38 C, aeration
rate 9 L/min, and
agitation of about 250 rpm. To this, 4.00 g of yeast was added.
When multiple enzyme¨metal systems were formed (the pH achieved 4.45, Fig.1),
the
solution of sodium selenite (in 50 ml of distilled water) was added (Table I).
During the
cultivation, ammonia hydroxide was added to adjust the pH to 4.6 - 4.7
(Fig.1). When the
fermentation was complete, the solution was centrifuged for 15 min (3500 rpm).
Then the yeast
was washed six times with water (10 times dilution) to remove water-soluble
residual selenium
compounds until negative reaction with SnC12 achieved and then was freeze
dried. The results
are shown in the Table I.
Table!.
Example 1 Example 2
Na2Se03 added to the fermenter, g 0.65 1.00
Yeast production (after freeze drying), g 11.83 10.64
Se content in dried yeast, ppm 3164 5040

CA 02649145 2012-12-18
12
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MIROSHNYCHENKO, OLEKSANDR
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MIROSHNYCHENKO, ZORYNA
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