Dietary supplementation with, and methods for administration of yeast-derived selenium product

The present invention solves the need for non-toxic forms of selenium which is an essential part of the human diet. This invention provides novel dried-yeast products containing selenium as well as a method of producing the dried yeast products. The method uses selenium having high biological activity but low toxicity. The invention also provides nutritional supplements containing the novel selenium containing dried yeast products and methods of administering these products and supplements to improve human health. The invention also provides a practically non-toxic yeast selenium product having increased intracellular selenium concentrations and methods to reduce tumor cell growth by administration of a selenium yeast product comprising yeast Saccharomyces boulardii sequela PY 31 (ATCC 74366) in combination with chemotherapeutic agents.

BACKGROUND OF THE INVENTION
 1. Field of the Invention
 This invention relates to the field of human dietary supplements and more
 specifically to improved therapeutic products prepared from yeast and
 containing increased selenium concentrations with reduced toxicities.
 2. Background
 Selenium is an essential trace element for proper physiological function in
 humans. Decades ago, scientists demonstrated that selenium was
 incorporated into the chemical structure of an enzyme called glutathione
 peroxidase, an enzyme that is necessary to protect erythrocyte (red blood
 cell) cell membranes, and other biological structures against toxic
 reactions with highly reactive oxygen-derived species such as peroxides,
 and superoxides. The role of selenium in the biochemistry of glutathione
 peroxidase has been studied in some detail and the most current medical
 information confirms that trace amounts of selenium are required to
 maintain normal human health. Failure to ingest and absorb the necessary
 amounts of selenium can lead to improper functioning of the body's
 metabolic processes, and to various diseases and disorders.
 Although many traditional nutrients, such as the natural occurring vitamins
 and minerals for which the U.S. Food and Drug Administration (FDA) has
 established a Recommended Daily Allowance (RDA), may be consumed in large
 quantities without adverse health effects, ingestion of high levels of
 some essential nutrients, such as certain metallic nutrients like
 selenium, may be toxic. To maintain ordinary health, one must balance the
 need for a minimum amount of such compounds with the need to protect
 against over-ingestion to the point of toxicity. For these compounds,
 ingesting low doses confers a significant nutritional benefit. However,
 when higher levels, i.e., amounts beyond the concentrations recognized as
 required for ordinary nutritional benefits, are ingested, beneficial
 health effects are not realized and the potential for dangerous toxicity
 exists.
 Nutritionally beneficial quantities for daily doses for selenium have been
 found to be small. Nutritional selenium levels have been established by
 the FDA (see 21 C.F.R. 101.9(c)(8)(iv), January 1994). Humans and animals
 can safely metabolize limited amounts of both inorganic and organic forms
 of selenium and can convert non-methylated selenium to mono-ordi-or
 trimethylated derivatives, of which the monomethylated derivatives are
 most toxic. [Bedwal, R. S., et al., Medical Hypotheses, 41(2):150-159
 (August 1993)]. The FDA has adopted Reference Daily Intakes (RDIs) of 70
 micrograms for selenium. Selenium dosage of 600 micrograms per day has
 been reported as safe. [Ferris G. M. Lloyd, et al., App. Clin.
 Biochem.,26:83-88 (1989)]. At about this dosage, normal activity of the
 enzyme glutathione reductase safely converts selenogluthatione to hydrogen
 selenide in the liver and erythrocytes and is ultimately excreted. Thus,
 at such lower dosages, the body is able to safely metabolize and excrete
 selenium that is present in the free metallic form.
 For many years, physicians and medical researchers have studied several
 potential health benefits resulting from the ingestion of low levels of
 selenium. For example, low concentrations of sodium selenate (an inorganic
 form of selenium) work with vanadium to improve glucose tolerance and to
 increase the levels of glucose-induced insulin release. However, as with
 many trace elements such as selenium, at higher dosage levels or
 concentrations, these beneficial effects are reversed and dangerous
 toxicity is manifested. [Furnsinn, C. et al., Internat'l J. of Obesity and
 Related Metab. Dis., 19(7):458-463 (1995)].
 Therefore, the administration of selenium in the natural form involves a
 scientific and medical trade-off because, when administered in relatively
 low concentrations, selenium provides beneficial health effects, however,
 at higher concentrations, selenium exhibits dramatic toxicity such that
 the potential health benefits are lost and toxicity becomes the primary
 concern. This trade-off is particularly problematic when selenium
 administration is attempted, not as a dietary supplement, but rather in
 the treatment of disease. However, if the toxicity problems could be
 overcome, increased dosages of selenium could offer substantial advances
 in the treatment of several important disorders that affect human health.
 For example, the role of selenium in maintaining the function of the
 enzyme glutathione peroxidase has led researchers to examine the role of
 selenium in several disease states. In cancer, animal studies have shown
 that selenium protected against chemicals and ultraviolet energy sources
 known to cause cancer in humans. Selenium is believed to reduce the risk
 of certain cancers due to its properties as a strong antioxidant. A
 clinical study of more than 1,300 people found that those who took a daily
 supplement of selenium cut their overall cancer risk by nearly 40%.
 [Terence Monmaney, Selenium May Fight Cancer, Study Shows, LOS ANGELES
 TIMES, Dec. 25, 1996, at A 1, A 29]. In addition, U.S. Pat. No. 4,599,234
 teaches that a combination of a selenium species (either organic or
 inorganic forms) with beta-carotene and a hydroxytoluene source
 significantly reduced the mortality of mice that were fed carcinogens and
 that these effects were better than those observed for the mice that were
 administered either selenium or beta-carotene or hydroxytoluene. U.S. Pat.
 No. 4,564,634 teaches a selenium-based nutritive composition having
 anti-neoplastic activity in which the selenium compound is used is a novel
 form of selenium prepared by a reaction of selenium metal with Tung oil
 (9,11,13-octadecatrienoic acid). [Schrauzer, G., Inorg. and Nutr. Aspects
 of Cancer, p. 330 (New York: Plenum Press (1978))].
 Heart disease has also been shown to be reduced in persons who consume
 recommended amounts of selenium in their diet. In certain studies, the
 levels of selenium in the blood stream have been directly correlated with
 the degree of progression of cardiovascular disease with those patients
 having the lowest levels of selenium having the most extensive coronary
 artery blockage. In such cases, the glutathione peroxidase enzyme is
 thought to exert an antioxidant effect that protects the coronary vessels
 from disease. In a similar mechanism, selenium is thought to interact with
 prostaglandins to control free radical cascades that lead to elevated
 levels of prostaglandins and inflammation. Patients suffering from
 arthritis have been shown to have low levels of plasma selenium and their
 clinical condition has improved with dietary supplementation of selenium.
 The precise mechanism by which selenium may protect from cardiovascular
 disease is not known, however, free radical antioxidant "scavengers," such
 as selenium, are believed to react with oxidants such that the oxidants
 are not available to form oxidized low density lipoproteins (O-LDLs).
 Thus, a reduction in the oxidants lowers the risk of arterial plaque
 deposits in blood vessels. Arterial plaque is precipitous material formed
 chiefly of oxidized low density lipoproteins (O-LDLs). The buildup of
 plaque in the form of O-LDL in the arteries is understood to be a factor
 in ischaemic heart disease. Free radical oxidants, many of which come from
 naturally occurring sources such as sun exposure, metabolism of certain
 nutrients, and exercise, act to oxidize low density lipoprotein (LDL) into
 its deleterious form, O-LDL. In contrast, high density lipoprotein (HDL)
 is understood to have beneficial health effects in the body. HDL is
 understood to be a more soluble form of lipoprotein, and its presence is
 not known to significantly contribute to the formation of arterial plaque.
 Since selenium functions to reduce the levels of O-LDL and thereby
 increase the level of HDL in the body, adequate quantities of selenium may
 decrease the likelihood of cardiovascular disease as well.
 Based on the foregoing, increased concentrations of selenium are potential
 treatments for a variety of disorders as long as the selenium
 concentrations do not reach toxic levels. For this reason, several
 different forms of selenium have been investigated to determine the
 optimal form for administration to humans, either as a dietary supplement
 or as a therapeutic product for the treatment of disease. Yeast-derived
 selenium has been shown to be a less toxic form of selenium, and thus a
 preferred source of a selenium composition for human consumption. The
 selenium produced by yeast cultures undergoes a type of biosynthesis
 whereby inorganic selenium salts are converted to an organic form via
 intracellular incorporation into the yeast. These organic, biosynthesized
 selenium yeast derivatives are better nutritive sources of selenium
 because they are less toxic and more easily metabolized by the mammalian
 system than their inorganic counterparts. One method of producing a
 selenium-enriched product using food yeast such as Saccharomyces
 cerevisiae or Candida utilis has been reported. When dried and fed to
 rats, these selenium-enriched yeast effectively prevent hepatic liver
 necrosis. [Reed et al., Yeast Tech., AVI Publ. Co., Conn. (1973)].
 Unfortunately, this method results in the production of a yeast product
 having a low intracellular selenium content, as well as a relatively high
 extracellular concentration of inorganic selenium.
 Generally, high extracellular concentrations of selenium are to be avoided,
 while higher intracellular concentrations are preferred because this tends
 to indicate an increased relative concentration of selenium in the organic
 form which, as noted above, is preferred for administration to humans. For
 this reason, prior efforts at producing selenium-based yeast products have
 focused on the ability to provide increased intracellular concentrations
 of selenium. For example, U.S. Pat. No. 4,530,846 ('846) describes a
 method for producing a selenium-enriched yeast that yields yeast with a
 moderately high intracellular selenium content. The yeast produced by this
 method are cultivated using a procedure that involves incremental feeding
 of the yeast culture. With respect to the process of the '846 Patent and
 the limitations on selenium concentration using that method, the '846
 patent states: "While intracellular selenium contents of yeasts are
 preferably in a 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." In
 addition to the recognized limitations on the ability to achieve higher
 concentrations of intracellular selenium, the prior art also demonstrates
 that the existing yeast-derived selenium products still exhibit
 substantial toxicity. For example, the LD.sub.50 for the yeast product
 described in the '846 patent is reported by the assignee to be on the
 order of 7 mg per kilogram. In practice, the LD.sub.50 rating for a
 product limits the amount that may be administered to a human as part of a
 nutritional program or as part of an overall therapy to treat a disease. A
 relatively high LD.sub.50 is particularly disadvantageous when physicians
 or researchers attempt to administer an elevated selenium dosage, i.e.,
 several times that recommended for dietary supplementation, in the
 treatment of disease.
 There remains a need in the art for a yeast selenium product that provides
 high concentrations of selenium, preferably the organic form fixed in an
 intracellular form, that exhibits the lowest possible toxicities when
 measured by LD.sub.50. Ideally, such a product would be provided by a
 method to produce selenium-enriched yeast that results in: (1) a high
 growth rate of selenium-enriched yeast; (2) selenium-enriched yeast with
 high intracellular selenium content; and (3) low toxicity.
 SUMMARY OF THE INVENTION
 The present invention overcomes the shortcomings of the prior art by
 providing a method for producing a composition of highly active
 nutritional selenium, where (1) the selenium source of the compositions of
 the present invention is the natural form of biosynthesized selenium; (2)
 the biosynthesized selenium is entirely metabolizable by the human system,
 and is substantially free of toxic substances; and (3) the process can be
 carried out efficiently and meets requirements important for commercial
 production.
 Therefore, an object of the present invention is to provide a method for
 preparing biosynthesized selenium-yeast with high selenium activity and
 low toxicity.
 It is a further object of the present invention to provide a method for the
 production of biosynthesized selenium having high nutritional value and
 low toxicity.
 Another object of the present invention is to provide an improved synthetic
 form of nutritional selenium that is substantially similar to the
 naturally occurring selenium complexes found in selenium rich foods.
 It is another object of the present invention to-provide a method for the
 production of selenium-enriched yeast, where the source of the yeast for
 metabolizing selenium substrate is a yeast strain such as Saccharomyces
 cerevisae or Saccharomyces boulardii sequela PY31.
 It is a further object of the present invention to provide a form of
 selenium species that is essentially non-toxic to the human body.
 It is another object of the present invention to-provide for methods for
 the treatment of disease by administering yeast-derived selenium in
 quantities greater than administered for dietary supplementation.
 It is a further object of the present invention to provide nutritional
 supplements that incorporate the selenium-enriched yeast produced by these
 methods.
 Another object of the present invention is to provide an increased dosage
 of selenium that is less toxic to the human body at higher dosages such
 that higher dosages can be administered, if desired, including the
 administration of yeast selenium with chemotherapeutic agents.
 DETAILED DESCRIPTION OF THE INVENTION
 The present invention relates to methods of cultivating yeast using
 selenium compounds resulting in a dried selenium-enriched yeast product
 with high biological activity, nutritional supplements comprising this
 dried yeast product, therapeutic products comprising this dried yeast
 product, uses of such dried yeast product to supplement the human diet,
 and methods to treat disease comprising administering dosages of selenium
 in combination with chemotherapeutic agents to reduce the growth of tumor
 cells.
 The process for preparing the selenium-enriched yeast product that has a
 high intracellular content of organically bound trivalent selenium in a
 highly biologically active and non-toxic form comprises the steps of:
 (1) preparing an aqueous mixture of yeast growth nutrients (aqueous media);
 (2) preparing an aqueous solution of selenium salt in distilled water by
 dissolving the selenium salt in warm distilled water and then filtering
 the resulting selenium solution;
 (3) adding the selenium solution to the yeast growth nutrients and mixing
 to form a selenium growth mixture;
 (4) adding the selenium growth mixture, preferably by incremental addition,
 to a live yeast culture to form a selenium yeast growth solution and
 incubating with gentle shaking action or stirring;
 (5) recovering and concentrating the yeast cells from the selenium yeast
 growth solution;
 (6) washing the recovered yeast cells to remove extracellular selenium; and
 (7) pasteurizing and/or drying the washed yeast cells.
 Growth media that can be used in the first- preparing step of present
 invention include 25.degree. Brix molasses [TCT, Gold Coast], 38.degree.
 Brix molasses [TCT, Gold Coast], glucose media, and potato dextrose broth.
 In addition, Brix molasses with a higher sugar content, e.g., 79.degree.
 Brix [TCT, Gold Coast], may be used and then diluted with distilled water
 to form a Brix solution of lower sugar content. Numerous other growth
 media that are known to support the growth of yeast from the Saccharomyces
 family may be used and could be readily selected by one of skill in the
 art. In a preferred form, a mixture of different growth media may be used.
 In addition, numerous vitamins and minerals may optionally be added to the
 yeast growth media. Such-vitamins and minerals are selected from those
 known in the art to help sustain proper yeast growth, including but not
 limited to biotin, vitamin B.sub.1, vitamin B.sub.6, calcium
 pantothenoate, inositol, copper, copper sulfate, zinc, zinc sulfate, iron,
 and iron sulfate.
 The second preparing step in the process for producing the
 selenium-enriched yeast of the present invention involves preparing a
 selenium solution by dissolving selenium salt in distilled water and
 filtering the resultant selenium solution. The selenium may be in the form
 of an amorphous solid or an organoselenium compound. In a preferred form,
 sodium selenite may be used. In a preferred form, a cellulose acetate
 filter [Coming Scientific Co.] may be used. The resultant filtered
 selenium solution has between about 100 ppm and 40,000 ppm selenium.
 The first addition step involves adding the selenium solution to the yeast
 growth nutrients (aqueous media) to form a selenium growth mixture. Once
 the selenium solution is added, the selenium and media may be mixed by
 gentle shaking action or stirring for between about 1 and about 30
 minutes.
 In the second addition step, the selenium growth mixture is added to live
 yeast cells to make a selenium yeast growth solution having selenium
 levels between about 100 ppm and about 20,000 ppm selenium, preferably
 between about 200 ppm to about 10,000 ppm, and most preferably between
 about 250 ppm to about 1,500 ppm of selenium. The second addition step
 preferably involves adding the selenium growth mixture to the yeast
 culture incrementally. This second addition step preferably takes place
 under a controlled pH of from about 4.2 to about 6.0, and preferably from
 about 4.5 to 5.3. This second addition step also preferably takes place at
 a temperature from about 20.degree. C. to About 35.degree. C., and
 preferably about 28.degree. C. to about 32.degree. C.
 The yeast employed in the second addition step preferably a food grade or
 edible yeast, and most preferably Saccharomyces boulardii sequela PY31.
 Other yeast which can be used include Saccharomyces Cerevisae or
 Saccharomyces Torula.
 As stated above, the present invention may also employ a newly isolated and
 purified strain of yeast, Saccharomyces boulardii sequela PY31.
 Specifically, Saccharomyces cerevisiae and Saccharomyces boulardii sequela
 are of the same genus, and Saccharomyces boulardii sequela is described as
 a synonym of Saccharomyces cerevisiae. [Barnett et al., Yeasts:
 Characteristics and Identification, Cambridge Univ. Press (1990)]. More
 particularly, the novel yeast strain Saccharomyces boulardii sequela PY31
 may be isolated from raw soil samples, and cultivated to yield quantities
 of yeast at a scale sufficient for developmental research and for
 production of commercial products. The novel strain of yeast,
 Saccharomyces boulardii sequela PY31, has been deposited in an
 International Repository in accord with the Budapest Treaty and has been
 assigned ATCC No. 74,366 ATCC American Type Culture Collection, 10801
 University Boulevard, Manassas, Va. 20110-2209. This novel yeast strain is
 described in co-pending application Ser. No. 08/719,572 filed on Sep. 25,
 1996. This strain may be analyzed according to the principle of cellular
 fatty acid analysis, to obtain a quantitative measurement of relatedness
 to Saccharomyces cerevisae based on their fatty acid make-up. A Euclidian
 Distance of less than 2.5 indicates that two yeast samples are most likely
 the same strain. A Euclidian Distance of less than 6.0 indicates the
 isolates are most likely the same subspecies and a Euclidian Distance of
 less than 10.0 indicates the isolates are most likely the same species.
 The greater the distance between the isolates, the closer the relationship
 taxonomically. In the present invention, the Euclidian Distance between
 the submitted strain (Saccharomyces boulardii PY-3-1) and LIBR S.
 cerevisae is 7.48, which indicates that these isolates are most likely the
 same subspecies, but the submitted strain is not very close to LIBR S.
 cerevisae due to the remote Euclidian Distance.
 Specifically, the method for isolating this novel yeast strain,
 Saccharomyces boulardii sequela PY31, comprises:
 (1) identifying a location for collection of a soil sample, which is
 proximal to a germanium mine (i.e., within 100 yards of a germanium mine);
 (2) sampling the soil by removing about 200 g from a depth of 5 cm to 20
 cm, and transporting the sample using a sterilized bag;
 (3) growing the living material on three different mediums which support
 the growth of all yeast, and that selectively kills bacteria without
 killing the yeast;
 (4) separating the yeast from other living matter and then repeating this
 process until yeast can be grown without bacterial contaminants;
 (5) selecting and restreaking the yeast colonies, and repeating this
 process three times;
 (6) selecting the yeast colonies most vital for growth in a medium enriched
 with germanium;
 (7) growing each selected colony on malt extract agar or dextrose agar, and
 selecting which colonies appear most robust, and;
 (8) cultivating the selected yeast by growing 1-2 slants of the yeast for
 about 2 days at about 30.degree. C. and then transferring to the
 cultivated yeast about 100 mL of malt extract broth and then incubating at
 about 30.degree. C. for 8-10 hours, then adding to the incubated mixture
 about 500 mL of malt extract broth and then growing the resulting mixture
 at about 30.degree. C. for about 6 to about 14 hours.
 The present invention also teaches a use of this novel yeast strain,
 Saccharomyces boulardii sequela PY31, to prepare selenium-enriched,
 non-toxic yeast forms according to the method described herein.
 As part of the second addition step, the selenium yeast growth solution is
 incubated to induce yeast growth. The incubation may occur with shaking or
 stirring at about 200 rpm for a period of about 5 hours to about 75 hours,
 preferably from about 15 hours to about 60 hours, and most preferably
 about 20 hours. This incubation occurs at a temperature of about
 25.degree. C. to about 30.degree. C., and preferably about 30.degree. C.
 The yeast cells are then isolated from the selenium yeast growth solution
 by centrifuging the selenium yeast growth solution, and isolating the
 yeast cells. In a preferred form, the centrifugation step may occur at
 about 3,900 rpm.
 The isolated yeast cells are then washed to remove extracellular selenium.
 The washing step may involve washing the isolated yeast cells between 2
 and 20 times with aqueous solvent, such as a buffered aqueous solution
 that optionally contains chelating agents such as EDTA.
 Lastly, the yeast cells are pasteurized and/or dried to produce a dried
 yeast product. The pasteurization step may occur at between about
 30.degree. C. and about 110.degree. C., preferably at about 60.degree. C.
 The resulting dried yeast product may contain from about 300 ppm to about
 6,000 ppm intracellular selenium, but preferably contains more than 1000
 ppm intracellular selenium, and most preferably between about 2000 ppm and
 about 5000 ppm.
 The present invention also relates to the use of the dried
 selenium-enriched yeast products as dietary supplements. To prepare the
 yeast compositions of the invention for use as a dietary supplement, the
 dried yeast product is combined as the active ingredient in intimate
 admixture with a suitable carrier according to conventional compounding
 techniques. This carrier may take a wide variety of forms depending upon
 the form of preparation desired for administration, e.g., oral,
 sublingual, nasal, or parenteral.
 In preparing the compositions in oral dosage form, any of the usual
 pharmaceutical media may be employed. For oral liquid preparations (e.g.,
 suspensions, elixirs, and solutions), media containing for example, water,
 oils, alcohols, flavoring agents, preservatives, coloring agents and the
 like may be used. Carriers such as starches, sugars, diluents, granulating
 agents, lubricants, binders, disintegrating agents, and the like may be
 used to prepare oral solids (e.g., powders, capsules, pills, and tablets).
 Controlled release forms may also be used. Because of their ease in
 administration, tablets, pills, and capsules represent advantageous oral
 dosage unit forms, in which cases solid pharmaceutical carriers are
 obviously employed. If desired, tablets may be sugar coated or enteric
 coated by standard techniques.
 For parenteral products the carrier will usually comprise sterile water,
 although other ingredients may be included, e.g., to aid solubility or for
 preservation purposes. Injectable suspensions may also be prepared, in
 which case appropriate liquid carriers, suspending agents, adjuvants, and
 the like may be employed.
 For dietary supplementation, a composition of the present invention is
 generally effective when parenterally administered in amounts ranging from
 about 1 mg of dried yeast per dose (1 dose per body weight of about 75 kg)
 to about 200 mg/dose of composition. A preferred amount is from about 10
 mg/dose to about 50 mg/dose, and most preferred at about 20 mg/dose. This
 dosage of the composition translates to an amount of from about 1
 .mu.g/dose to about 200 .mu.g/dose of selenium, preferably from about 10
 .mu.g/dose to about 50 .mu.g/dose of selenium, and most preferably about
 20 .mu.g/dose of selenium. When orally administered, the compositions of
 the present invention are generally effective in approximately the same
 amounts as the parenteral products. Activity at this level makes the
 compositions particularly well suited for formulations in tablet size for
 oral administration. The above dosage ranges are likely to be administered
 at varying periods for humans, for example, from daily administration to
 administration at least 5 times per week. However, ultimately, the dosage
 regimen will depend upon the particular needs of the user. A preferred
 dosage regimen for dietary supplementation in humans is 1-2 doses per day.

The following examples are illustrative only and do not limit the invention
 in any fashion. Examples 1-3 demonstrate a process to yield a selenium
 yeast product having in intracellular yeast concentration of greater than
 1000, greater than 1800 and approximately 2000 ppm.
 EXAMPLE 1
 The growth medium was prepared as follows. 79.degree. Brix molasses (1000
 g) from TCT, Gold Coast was diluted to 1 L with distilled water resulting
 in a 32.degree. Brix solution. Then, 4.12 g of KCl was added, followed by
 4.12 g of MgSO.sub.4.7H2O and then 44.4 g of NH.sub.4 HPO.sub.4, and the
 resulting mixture was stirred to homogeneity. To this mixture was added
 enough water to reach a final volume of 2 L, and thus a 32.degree. Brix
 molasses. The mixture was tested for sugar content by using a Brix
 refractometer [Cole-Palmer Instrument Co., RH13-32ATC], and the final pH
 was adjusted to 5.0 with HCl or NaOH. This solution was then autoclaved at
 105.degree. C. for 15 minutes. A stock solution of sodium selenate was
 prepared as follows. To 300 mL of distilled water at ambient temperature
 was added 9.0 g of sodium selenate and the resulting mixture was kept at
 ambient temperature for 1 hour, then filtered through cellulose acetate
 membrane [Coming Scientific Co].
 The selenium growth mixture was prepared by adding 60 mL of the stock
 selenium solution to 1300 mL of 32.degree. Brix molasses mixture (pH 5.07)
 and 3140 mL of water.
 The yeast culture was prepared as follows. One slant of yeast were
 incubated for two days at 30.degree. C., and then grown at 30.degree. C.
 in 200 mL of Malt extract, shaken at 200 rpm for 8 hrs, and then to this
 was added 300 mL of malt extract and the resulting mixture was incubated
 at 30.degree. C. overnight with shaking.
 The yeast were cultivated as follows. 500 mL of a solution of Saccharomyces
 boulardii sequela PY31 was dissolved in 500 mL of distilled water and
 stirred at 500 rpm in a 7 L fermentation apparatus [Bioflow 2000, New
 Brunswick]. To this yeast culture, 4500 mL of the selenium growth mixture
 as added under an air flow of about 2 to about 5 L/min and over a period
 of about 6 hours at about 30.degree. C. The resulting suspension was
 stirred for an additional 15 hours.
 The resulting selenium yeast growth solution was then centrifuged at 3900
 rpm for 10 minutes, and resulting-yeast cream (isolated yeast cells) was
 washed five times with a total volume of 8 L of water.
 The resulting yeast cells were dried at less than 80.degree. C. until a
 moisture content of 2-3% was obtained. The selenium was kept at ambient
 temperature for 1 hour, then filtered through cellulose acetate membrane
 [Coming Scientific Co].
 The selenium growth mixture was prepared by adding 60 mL of the stock
 selenium solution to 1300 mL of 32.degree. Brix molasses 5 mixture (pH
 5.07) and 3140 mL of water.
 The yeast culture was prepared as follows. One slant of yeast were
 incubated for two days at 30.degree. C., and then grown at 30.degree. C.
 in 200 mL of Malt extract, shaken at 200 rpm for 8 hrs, and then to this
 was added 300 mL of malt extract and the resulting mixture was incubated
 at 30.degree. C. overnight with shaking.
 The yeast were cultivated as follows. 500 mL of a solution of Saccharomyces
 boulardii sequela PY31 was dissolved in 500 mL of distilled water and
 stirred at 500 rpm in a 7 L fermentation apparatus [Bioflow 2000, New
 Brunswick]. To this yeast cultures 4500 mL of the selenium growth mixture
 as added under an air flow of about 2 to about 5 L/min and over a period
 of about 6 hours at about 30.degree. C. The resulting suspension was
 stirred for an additional 15 hours.
 The resulting selenium yeast growth solution was then centrifuged at 3900
 rpm for 10 minutes, and resulting yeast cream (isolated yeast cells) was
 washed five times with a total volume of 8 L of water.
 The resulting yeast cells were dried at less than 80.degree. C. until a
 moisture content of 2-3% was obtained. The selenium content of the yeast
 was measured using atomic absorption techniques to give a yeast mass
 having 1839 ppm of selenium. As noted above and in the following examples,
 the concentration of intracellular selenium can be modified by adjusting
 the concentration of selenium in the stock solution of sodium selenate
 also, the process steps can be manipulated as described herein to alter
 the final selenium concentrations in the yeast product. The final
 intracellular selenium concentration is preferably greater than 1000 ppm,
 preferably greater than about 2000 ppm, and as high as about 5000 ppm and
 exhibits non-toxicity as measured by LD.sub.50 such that doses of the
 yeast product of the invention equivalent to greater than 7 mg/kg in rats,
 can be administered without toxicity, including amounts as high as 5.0
 g/kg or higher. The resulting selenium yeast product of the invention
 meets the requirements of the Limit Test as set forth in the
 "Toxicological Principle for the Safety Assessment of Direct Food
 Additives and Colour Additives Used in Food."
 EXAMPLE 2
 The method described in Example 1 was repeated to give a dried yeast mass
 having a final concentration of selenium of 1849 ppm.
 EXAMPLE 3
 Examples 3 and 4 describe a process to produce a yeast product having
 increased selenium concentrations, specifically greater than 4500 ppm and
 approximately 5000 ppm. The yeast growth nutrients were prepared as
 follows. 25.degree. Brix molasses (670 g) [TCT, Gold Coast] was diluted to
 1 L with distilled water, then 2.72 g of KCl was added, followed by 2.72 g
 of M9SO.sub.4.7H2O and then 29.28 g of NH4HPO.sub.4, and then enough water
 was added to reach a final volume of 2 L, and the resulting mixture
 stirred at ambient temperature until homogenous. The mixture was tested
 for sugar content by using a Brix refractometer [Cole-Palmer Instrument
 Co., RH13-32ATC], and the final pH was adjusted to 5.0. This solution was
 autoclaved at 105.degree. C. for 15 minutes.
 A stock solution (3%) of sodium selenate was prepared as follows. To 300 mL
 of distilled water at ambient temperature was added 9.0 g of sodium
 selenate and the resulting mixture was stirred at ambient temperature for
 1 hour. The resulting selenium solution was filtered through cellulose
 acetate membrane [Coming Scientific Co].
 The yeast culture was prepared as follows. One slant of yeast were
 incubated for two days at 30.degree. C., and then grown at 30.degree. C.
 in 200 mL of Malt extract, shaken at 200 rpm for 8 hrs, and then to this
 was added 300 mL of malt extract and the resulting mixture was incubated
 at 30.degree. C. overnight with shaking.
 A yeast growth mixture was prepared by adding 13.2 L of the selenium
 solution (3%) to 3989 mL of the 25.degree. Brix molasses and 247 mL of
 water, and then stirred or shaken to homogeneity.
 The cultivation of selenium-enriched yeast was undertaken as follows. In a
 7 L fermentation apparatus [Bioflow 2000, New Brunswick] containing 70 mL
 of a stirred (500 rpm) yeast culture of Saccharomyces boulardii sequela
 PY31, 2 L of the yeast growth nutrient mixture was added over 8 hours at
 30.degree. C. with stirring under an air flow of about 2 to about 5 L/min.
 After the addition of the yeast growth nutrient mixture, the mixture was
 stirred an additional 5 hours at about 500 rpm. Then, 3989 mL of
 25.degree. Brix molasses growth nutrients and 247 mL of water were added,
 and the resulting selenium yeast growth solution was shaken at 200 ppm for
 24 hours at 30.degree. C.
 The resulting selenium yeast growth solution was centrifuged at 3,900 rpm
 for 10 minutes, the supernatant removed, then the yeast cells were washed
 once with 100 mL of EDTA (pH=7.8), once with 100 mL of 0.01 M Na.sub.2
 HPO.sub.4 buffer solution, and five times with 100 mL of distilled water.
 The resulting yeast cream (isolated yeast cells) was dried in vacuo and
 then the selenium content of the yeast was measured using atomic
 absorption techniques to give a yeast mass having 4857 ppm of selenium.
 EXAMPLE 4
 The method of Example 3 was repeated and yielded a final dried yeast mass
 having a concentration of selenium of 4945 ppm.
 EXAMPLE 5
 A 20% glucose media was prepared by adding 400 g of glucose, with stirring,
 to 2 L of distilled water, followed by addition of 43 g of urea, 20 g of
 Na.sub.2 HPO.sub.4, 7.6 g of MgSO.sub.4.7H.sub.2 O, 44 g of KCl, 50 g of
 sodium citrate, and 10 grams of yeast extract [DIFCO, Bacto]. The
 resulting yeast growth nutrients were mixed and then autoclaved at 10 psi
 for 15 minutes.
 A vitamin mixture was prepared as follows: 4 mg solid biotin, 8 mg vitamin
 B1, 200 mg vitamin B6, 100 mg calcium pantothenoate, and 2 g of inositol
 were added to 100 mL of distilled water, and the resultant solution was
 stirred to homogeneity and filtered through a 25 micron cellulose acetate
 filter [Watman].
 A mineral solution was prepared as follows: 0.5 g of CuSO4.5H.sub.2 O, 8 g
 of ZnSO.sub.4.7H.sub.2 O, and 3 g ferric sulfate (Fe(SO.sub.4).sub.2
 (NH.sub.4).sub.2.6H.sub.2 O) were added to 1 L of distilled water and
 stirred to homogeneity and then autoclaved at 10 psi for 15 minutes.
 A yeast growth nutrient mixture was prepared by adding 20 mL of the vitamin
 solution and 2 mL of the mineral solution to the glucose media.
 The yeast culture was prepared as follows. One slant of yeast were
 incubated for two days at 30.degree. C., and then grown at 30.degree. C.
 in 200 mL of Malt extract, shaken at 200 rpm for 8 hrs, and then to this
 was added 300 mL of malt extract and the resulting mixture was incubated
 at 30.degree. C. overnight with shaking.
 A cultivation of selenium-enriched yeast was undertaken by adding 50 mL of
 3% sodium selenate solution (as described in the above examples) to 2,500
 mL of the 20% yeast growth nutrient mixture. Next, 2 L of the resulting
 mixture was isolated and added slowly over a period of 11 hours at 31
 .degree. C. to a mixture of 500 mL Saccharomyces boulardii sequela PY31
 (ATCC No. 74,366) and 2 L distilled water that had been mixed to
 homogeneity and autoclaved at 10 psi for 15 minutes. The resulting
 selenium yeast growth solution was then stirred at 31.degree. C. for an
 additional 8 hours.
 The yeast were then isolated as described in Example 1 above to yield yeast
 mass containing 684 ppm selenium.
 EXAMPLE 6
 A determination of toxicity of the yeast product of the present invention
 was conducted in the Rat Acute Oral Toxicity Model for comparison with the
 reported values in existing selenium yeast products and specifically for
 those described in U.S. Pat. No. 4,530,846 (LD.sub.50 indicates toxicity
 at 7 mg/kg). The Rat Acute Oral Toxicity Study was carried out on the
 yeast selenium product of the invention at 1.0, 2.0, 3.0 and 6.0 g/kg body
 weight. Groups consisted of 3 male rats per dose level. No mortalities or
 toxic symptoms were observed at any of these dose levels.
 Based on the results of this toxicity testing, a limit test was performed
 using one dose level of 5.0 g/kg body weight. The test article was
 suspended in 1% methylcellulose (w/v.) 15.0 g of the test article was
 suspended in 60.0 mL: of 1% methylcellulose and thoroughly blended by use
 of a Polytron.RTM. homogenizer. The test article was administered through
 a feeding cannula at a dose level (by volume) of 20.0 mL/kg. The test
 group consisted of 5 male and 5 female rats. No mortalities or other toxic
 symptoms were observed during the 14-day study period, and all animals
 gained body weight by the end of the study. No gross pathological findings
 were observed at the end of the study.
 Based on the above findings, the yeast selenium product of this invention
 is classified as "practically non-toxic" by oral ingestion and the
 LD.sub.50 for the product was determined to be in excess of 5.0 g/kg.
 EXAMPLE 7
 As noted above, the administration of a selenium yeast product can be
 indicated in the treatment of disease. Because the selenium yeast product
 of this invention demonstrates low toxicity, the selenium yeast product
 can be readily administered with other therapeutic compounds including
 chemotherapeutic agents to reduce the growth of tumor cells. For example,
 the anti-neoplastic effect of the selenium yeast product of the invention
 have been investigated on the following tumor cells in vitro; Breast
 (MCF-7, MCF-10, SKBR-3), Lung (RH.sub.2), Prostate (LNCap and PC-3), Colon
 (T84, Caco-2), Small Intestine (HCF8), and Liver (HepG2). In addition, we
 examined additive or synergistic effect of the selenium yeast product in
 combination with standard anti-cancer drugs, adriamycin and taxol. The
 effect of the administration of the selenium yeast product was assessed
 with the following measurements: apoptosis; DNA synthesis; growth rate by
 MTT assay; uptake of amino acid MeAIB by System A; and morphological
 changes by the tunnel assay. The selenium yeast caused increase in
 apoptosis as measured by DNA fragmentation and increase in "rounded" cells
 and membrane "blebbing," decrease in MeAIB uptake, and decrease in DNA
 synthesis in breast cancer cells MCF-7 and SKBR-3. These changes were
 selenium dose dependent with optimal inhibition at a selenium
 concentration between 4 and 40 ng/ml after 72 hrs of treatment. Similar
 observations were with lung (RH2), small intestine (HCF8), colon (Caco-2),
 and liver (HepG2) cells. In contrast, prostate cancer cells LNCap and
 PC-3, and the colon cancers cells T-84 were not significantly affected by
 administration of the selenium yeast product alone. However, addition of
 adriamycin or taxol in combination with selenium inhibited growth of
 prostate cancer cells. Addition of a chemotherapeutic agent, in this case,
 taxol or adriamycin, with the selenium product caused further inhibition
 of MCF-7, SKBR-3, RH2, HCF8, and HepG2 cells. Thus, the selenium yeast
 product had a significant in vitro anti-neoplastic effect on breast, lung,
 liver, and small intestinal tumor cells. Supplementation of the
 anti-neoplastic therapy with the selenium yeast product potentiated
 chemotherapeutic effects of taxol and adriamycin in the cancer cell lines
 indicated above. Thus, a preferred method of this invention is the
 administration of a selenium yeast product in combination with a
 chemotherapeutic agent such as Adriamycin, Doxil, Mitoxantrone, Mitomycin
 C, Actinomycin, Cytoxan, Bleomycin, BCNA (Carmustine), Velban,
 Vincristine, Epotoside, Prednisone, Xeloda, S-Fluorouracil, Camptosar,
 Carboplatin, Cis-Platin, Taxol, Taxatere, Herceptin, Methotrexate,
 Hycroxyurea, Cytosar, Gemcytabine, Nitrogen Mustard, Procarbazine,
 Imidazove Carboxamide (Dacarbaziin), Tamoxifen, Estracyte, Jupron,
 Flutamide, Daunarubicin, Lomostine, Ifusfamide, Mesna, Streptozocin,
 Interferon, Dexamethasone, Melphalan, Leulovorin, Chlorambucil,
 Fludarabine, Busulfan, Interleurine-2, Navelbine, Gmerocapto-Purina, and
 Megestroc.
 EXAMPLE 8
 As noted above, the yeast products of the invention exhibit significantly
 lower toxicities compared to other selenium yeast products manufactured
 with existing techniquest and with non-proprietary yeast strains such as
 ordinary Saccharomyces cerevisae (Brewer's Yeast).
 To compare the advantage of the yeast strains described herein with
 ordinary S. cerevisae, about 1 g of each yeast strain sample (a total of
 10 samples were collected) was added to 99 ml of malt broth (pH 4.7,
 DIFCO), shaken at 200 rpm at 30.degree. C. for 2 days. The fungal conidia
 germinate and form small mycelia balls that do not form new conidia (Rose
 et al., 1987). After two days of growth, the culture solution was filtered
 though the sterilized glass wool and the fungal mycelium could be filtered
 off over the wool. Serial dilution (10 fold) of the filtrate were made and
 then streaked onto three types of agars with 0.1% chlorophenicol or
 ampicillin (50 ug/L), including malt extract agar (pH 4.7, DIFCO),
 sabouraud dextrose agar (pH5.6, DIFCO), and potato dextrose agar (pH 5.6,
 DIFCO). The colonies were selected under microscope and restreaked after
 2-7 days of incubation at 28.degree. C. This procedure was repeated at
 least 3 times.
 Yeast stocks were maintained at 30.degree. C. on malt agar (DIFCO) or
 sabouraud dextrose agar (DIFCO). For most experiments, cultures were grown
 in a molasses medium had the following composition (g L-1): 79.degree.
 Brix molasses JCT, Gold Coast, Calif.), 80.8; KCI, 0.33; MgSO4, 0.33;
 NH4H2PO4, 3.6; pH 4.5-5.0. pH was adjusted to 4.5-5.0 using HCI. The
 cultures were allowed to grow to A 590=(log phase, 1.2-2.4.times.10.sup.6
 cell/ml) prior to use. Cell numbers and percentage of budding cells and
 dead cells were determined using a haemocytometer after appropriate
 dilution with distilled water and staining with 0.4% trypan blue solution
 (sigma, St, Louis, Mo.). The size of yeast populations in aqueous
 suspensions was determined using optical density (OD590) measurements.
 The yeast strains (YP3-1 Saccharomyces boulardii Sequela, ATCC 9763 and
 ATCC 96473), were allowed to grow to 1-2.times.10.sup.6 cell/ml in the
 malt broth medium, followed by incrementally feeding with molasses (Gold
 Coast, Calif.) and sodium selenate (Sigma, St. Louis, Mo.). The final
 resulting mixture containing 300-400 ppm of sodium selenate and
 5-6.degree. Brix of molasses was incubated at shaker (260 rpm, 30.degree.
 C.) or at the fermentor (BIOFLO 2000 FERMENTOR, New Brunswick Scientific,
 Edison, N.J.) at 600 rpm, 3 L/min and 30 T. After 21-27 hr of incubation,
 this mixture was centrifuged at 3,900 rpm for 10 minutes, the supernatant
 removed, then the yeast cells were washed once with 0.1 M EDTA (pH=7.8)
 and 0.01 M Na.sub.2 HPO.sub.4 buffer solution, and then five times with
 distilled water. The resulting yeast cream was dried in 80.degree. C. oven
 for 2-3 days.
 The selenium concentration in Se-yeast product was measured using an atomic
 absorption spectrometer (Perkin Elmer, Norwalk, Conn., Model 3100). To
 accomplish this measurement, the dried yeast sample was digested in
 concentrated HNO.sub.3 for four hours under a hood. Then, its selenium
 content was measured using an atomic absorption spectrometer set at a
 wavelength of 196.0 nm and a selenium lamp current at 16 mA.
 The yeast strains were selected based on its superior vitality when grown
 in the presence of 1 ml aliquots of growing yeast culture containing 200,
 300, 400, and 500 ppm of sodium selenate. After incubation at 30.degree.
 C. for 1 day, the culture was streaked on malt extract agar, potato
 dextrose agar and sabouraud dextrose agar. The yeast (YP 3-1), gobose and
 budding cells, was growing well in solution containing sodium selenate
 under the concentration of 400 ppm and was selected from sabouraud
 dextrose agar with ampicillin. The colony characteristics of this yeast
 were described as dull creamy, non- mucoid, slight raised colonies on YM
 agar. ATCC 9763 and ATCC 96473) were commercially obtained from ATCC.
 Table 1 and table 2 show the comparison of three strains in Se uptake
 ability and yeast growth characteristics by shaker and BF 2000 fermentor.
 Both experiments show less dead cells, higher mass and higher selenium
 concentrations in YP3- 1 yeast product than that in control. In table 1,
 YP3-1 yeast produced 17.2% and 26.0% more dry mass containing 10.6% and
 7.1% more cellular selenium than that from ATCC 9763) and ATCC 96473
 yeast. More significantly in table 2, YP3-1 yeast produced 28.9% and 32.0%
 more dry mass containing 34.6% and 18.6% more cellular selenium than that
 from ATCC 9763 and ATCC 96473 yeast. The results indicate that selected
 yeast strain (YP31) has higher ability to assimilate selenium and shows
 efficient sugar utilization, higher toxic tolerance to selenium and better
 fermentation rate compared to the yeast strains tested.

Se
 concentration
 yeast quality dry weight in final Se-
 Yeast strain (after fermentation) (g) yeast (ppm)
 Invention 1.34 .times. 10.sup.8 cell/ml 2.15 2406
 3-1 33% budding
 Sgrula 1.1% dead cell
 ATCC 9763 4.44 .times. 10.sup.7 cell/ml 1.78 2150
 48.4% budding
 12.7% dead cell
 ATCC 96473 2.82 .times. 10.sup.7 cell/ml 1.59 2235
 41.3% budding
 3.75% dead cell
 Table 1. Se Accumulation of Different Yeast Strains in Shaker
 To 50 ml of yeast culture (YP 3- 1, ATCC 9763, and ATCC 96473) and 349 ml
 of dH20, 6.5 ml of 3% Sodium selenate and 95 ml of 32.degree. Brix
 molasses mix (pH 4.68) were incrementally fed. The mix (total 500 ml)
 containing 390 ppm Na2SeO4 compound, 6.degree. B molasses mix and 10%
 yeast culture was incubated in a shaker at 260 rpm, 30.degree. C. for 27
 hrs. The yeast recovery and drying process were as described above and the
 results are an average of 3 experiments.

Se
 wet solid concentration
 yeast quality weight weight in final Se-
 Yeast strain (after fermentation) (g) (g) yeast (ppm)
 3-1 3.41 .times. 10.sup.7 cell/ml 161 29.1 1418
 22.67% budding
 0.34% dead cell
 ATCC 9763 2.4 .times. 10.sup.7 cell/ml 119 20.7 928
 21.3% budding
 2.3% dead cell
 ATCC 96473 1.1 .times. 10.sup.7 cell/ml 114 19.80 1154
 10.32% budding
 0.45% dead cell
 Table 2. Se Accumulation of Different Yeast Strains in Fermentor
 To 500 ml of pure yeast culture (YP 3-1, ATCC 9763, and ATCC 96473) and
 3435 ml of dH.sub.2 O, 65 ml of 3% Sodium selenate, 150 ml of dH.sub.2 O
 and 853 ml of 32.degree. Brix molasses mix (pH 4.68) were incrementally
 fed to BF 2000 fermentor. The mix (total 5 L) containing 390 ppm sodium
 selenate compound, 5.45.degree. B molasses mix and 10% yeast culture was
 incubated in the fermentor at 600 rpm, 30.degree. C. for 21-21.5 hrs. The
 yeast recovery and drying process were as described above and the results
 are from the average of three duplicate experiments.
 Numerous modifications and variations of the present invention are included
 in the above-identified specification and are expected to be obvious to
 one of skill in the art. It is also intended that the present invention
 cover modifications and variations of the dried yeast selenium
 compositions and method for using them to accomplish their claimed uses
 within the scope of the appended claims and their equivalents.