Abstract:
A process for preparing selenium yeast having a high intracellular selenium content is provided which comprises the continuous incremental feeding of nutrients and selenium compounds to yeast during the growth cycle.

Description:
FIELD OF THE INVENTION 
     This invention relates to a method for the preparation of yeast containing substantial amounts of intracellular organically bound selenium, useful as a supplementary diet source of metabolizable selenium where additional selenium is indicated. 
     DESCRIPTION OF THE PRIOR ART 
     While the role of selenium in mammalian diets is not completely understood, certain investigative work has indicated that selenium supplemented diets can play a role in the prevention of certain conditions in mammals. For example, food yeast such as Saccharomyces cerevisiae and candida utilis which incorporate selenites during growth, when dried and fed to rats, effectively prevent hepatic liver necrosis. See, Reed et al., Yeast Technology AVI Publishing Co. (Conn. 1973) p. 41. More recently, experiments have been conducted indicating that organically bound selenium in the diets of mammalians can be a factor in prevention of cancer. 
     The use of selenium in mammalian diets is limited, however, by virtue of the fact that many inorganic and organic selenium compounds such as salts are highly toxic and cannot be consumed in substantial amounts by mammals in that form, particularly as inorganic compounds. It is known, however, that selenium compounds may be used as an additive component of the nutrient substrate for growing certain food yeasts such as baker&#39;s yeast or Saccharomyces cerevisiae to produce yeasts that contain intracellular selenium compounds in an organically bound ingestible form whose exact composition is not known. The organically bound intracellular selenium as it is found in yeast that has been grown in a selenium rich nutrient is readily assimilable and efficient as a dietary source of selenium in mammalian diets without exhibiting the normal toxic consequences associated with diets containing supplemental selenium compounds in other chemical forms particularly as inorganic selenium. 
     Inasmuch as there has been considerable interest in obtaining a form of selenium which can be introduced into the diet as a supplement in substantial quantities without the aforenoted toxic consequences, it would be desirable to produce a food source containing selenium and more particularly a food yeast which contains an intracellular non-toxic assimilable form of selenium to be used as a dietary supplement for mammals including humans. 
     It has been found that the introduction of selenium salts as a component of the nutrient substrate of yeasts produced by conventional batch processing results in a substantial amount of the selenium being absorbed by the yeast in an organic and biologically combined form that is both assimilable and non-toxic to mammals which subsequently ingest the selenium yeast as a dietary supplement. 
     The production of selenium yeast by such procedures, however, is limited by a number of factors. For example, high concentrations of selenium salts in the nutrient substrate used to grow the yeast has an inhibitory effect on the growth of yeast and consequently the ultimate yield of yeast (based on molasses) that may be obtained by that procedure. Furthermore the amount of selenium in the yeast recovered by the aforenoted batch yeast growth process in the desired organically bound form is relatively low. Finally, it has been determined that the selenium in recovered yeast produced by the aforenoted batch-feeding technique has an undesirably high potential free selenium toxicity risk as established by the Methylene Blue Reduction Test. 
     OBJECTS OF THE INVENTION 
     Accordingly, one of the objects of the present invention is the provision of a method for producing a selenium yeast having an enhanced intracellular selenium content. 
     Another object is the provision of a method for producing a food yeast having high levels of organically bound intracellular selenium in an assimilable non-toxic form. 
     A further object is the provision of a method and process for producing an edible food yeast product having a high intracellular selenium content which is useful as a dietary supplement. 
     A final object is the provision of a food yeast having a uniformly distributed intracellular selenium content of high concentration, but which is essentially free of the toxic risk of free selenium as established by the Methylene Blue Reduction Test. 
     SUMMARY OF THE INVENTION 
     The foregoing objects are achieved by a process and method for preparing an edible selenium yeast product which has a high intracellular, organically bound, assimilable selenium content, which process comprises continuously and incrementally feeding a substantial quantity of a soluble selenium salt to a food yeast such as Saccharomyces cerevisiae maintained in a growth medium under aerobic yeast growth conditions in conjunction with the incremental addition of a carbon nutrient source such as molasses wort, to said medium, to support the growth of the yeast. The growth medium is further characterized by low levels of inorganic sulfur nutrients. &#34;Low levels&#34; of inorganic sulfur means less than about 0.5% and preferably about 0.1% to 0.2% sulfur on a yeast solids basis. Broadly however, the inorganic sulfur content of the nutrient media should be maintained at sufficiently low levels so as not to substantially adversely affect the yield of yeast produced by the process. 
     After the growth cycle is completed, the yeast is harvested from the fermenter such as by centrifugation and washed essentially free of extracellular, water-soluble residual selenium compounds to produce an edible selenium yeast which is characterized by an intracellular organically bound selenium content of at least 1000 ppm and characterized by being essentially free of extracellular inorganic selenium as determined by the Methylene Blue Reduction Test. 
     Yeast products with intracellular selenium contents of 1000 ppm or more are achieved by the continuous incremental feeding of soluble selenium compounds to the nutrient growth media for the yeast at levels which are as high as 128 ppm on a fermenter volume basis or about 0.4% Se in the molasses which is incrementally fed to the fermenter. The selenium salt addition is conveniently carried out in the last or final fermenter stage of commercial yeast propagation, sometimes called the &#34;trade&#34; fermenter in the context of conventional commercial multi-batch yeast fermentation processes but may be carried in any isolated yeast growth fermentation procedure. 
     It should be understood that at any given time in the fermentation process, the concentration of selenium salt should not be as high as to inhibit the growth of the yeast. In this context, it has been found that periodic additions of selenium salts at selected intervals, i.e., at the 81/2, 9th, 91/2, and 10th, hours of fermentation at the high levels calculated to achieve an intracellular selenium content in the harvested yeast of about 1000 ppm or more, resulted in a reduction of the yeast yield (based on molasses) to levels of about 40% and additionally resulted in a lower level of intracellular selenium in the final yeast produced by that procedure. It has been found that incremental addition of the selenium salt throughout the yeast fermentation and growth process obviates these undesirable adverse results. The continuous incremental addition of the selenium salts or compounds can readily be carried out by mixing the selenium compounds with the molasses feed (wort) which is also continuously added to the yeast fermenter. This procedure usually requires temperatures of less than about 120° F. (45° C.) to avoid possible degrading of the selenium compound, e.g., sodium selenite. If the wort temperature used in the method are above about 120° F. (45° C.), then the selenium salts (if unstable at higher temperatures) are preferably added separately (as in a solution) as a continuous incremental feed to the fermenter simultaneously and concurrently with the addition of the carbon source nutrient throughout the entire fermentation period, e.g., over a time period of from 14 to 16 hours. 
     A typical commercial operation for producing yeast is described in Reed &amp; Peppler, Yeast Technology, pp. 79-80 (1973). That process starts in the laboratory, where Pasteur flasks containing a rich medium (malt extract or malt extract molasses blend) are inoculated from slants of the pure yeast culture. The contents of the Pasteur flasks, after typically two (2) to three (3) days incubation is then inoculated into small pure culture fermenters usually a series of three pure culture fermenters with capacities of, e.g., 20 gal., 100 gal., and 1000 gal. The yeast is grown in these fermenters in a sterile medium rich in growth factors. There is no incremental feeding of nutrients and/or molasses in this growth stage and little aeration. The air used is customarily sterilized. The last pure culture stage has been designated the (F1) stage. 
     The pure culture fermenter stages are followed by one or more incrementally fed stages (F2, F3, etc.). A portion of the fermenter contents of the (FI) stage is pumped into a larger tank for the first incrementally fed stage (F2). From this point on in the yeast propagation system, aeration is vigorous and molasses and other nutrients are fed incrementally. After completion of each of these incrementally fed stages of fermentation, the yeast is separated from the bulk of the fermenter liquid (beer) by centrifuging, producing a stock of yeast for the next stage. The fermenter contents of a completed incrementally fed stage is usually divided into several parts for pitching of the next stage of fermentation. After the final incrementallly fed stage of fermentation, also called the &#34;trade&#34; stage, the yeast is removed from the fermenter beer by centrifugation to produce a yeast cream of 13-16% solids and washed. In the present process, the yeast is washed up to as many as seven (7) to ten (10) times or as necessary to reduce or substantially remove the extracellular nutrients and extracellular selenium salts from the yeast by re-suspending the yeast in water (diluted in ratios of about 1:10 when washing is completed the yeast is reduced in the moisture content to a cream (16% yeast solids) followed by pasteurizing and drum drying so as to reduce its moisture content to about 4 to 5% to produce a dried yeast product. The pasteurization should be carried out at 85° C. for 45 minutes in cream stage or similar or equivalent conditions to non-viable yeast product. 
     Incremental feed, as used above, refers to the practice of adding the molasses and other nutrients to the fermenter liquid-yeast mixture preferably at such a rate that the molasses is consumed by the yeast at the same rate it is added. Since the yeast population increases with the growth cycle, the rate at which the nutrient added is consumed is also increased. In the process of this invention, the selenium salts are added continuously and incrementally as a separate feed or admixed with the molasses wort, by the technique discussed above. 
     The dried yeast is tested for extracellular inorganic selenium by the Methylene Blue Reduction Test (MBRT) which involves the following test procedure. 
     1. Place in vial, 0.5 g powder or sample containing approximately 100 μg Se in product. 
     2. Add 5 mls of &#34;Reducing Solution&#34; containing 20% solution of 1-thioglyercol in 0.2N phosphate buffer pH 5.5. 
     3. Shake closed vial for 10 seconds. 
     4. After 3 minutes, at t=0, add 2 drops of Methylene Blue solution containing 2% w/v Methylene Blue in doubly distilled water. 
     5. Close the vial and shake for 10 seconds. 
     6. Record time at which decolorization is essentially complete. This is referred to as MBRT time. 
     The time of test indicates the presence of inorganic selenium. More than fifteen (15) or twenty (20) minutes indicates a sufficiently low extracellular inorganic selenium content. For example, baker&#39;s yeast without any selenium will give an MBRT of ninety (90) to one hundred fifty (150) minutes. Yeasts with substantial extracellular selenium, e.g., 10 ppm. will give MBRT times of from 0.5 to 7 minutes. 
     In the present process the selenium salts used are water-soluble selenium compounds, preferably inorganic compounds of which sodium selenite (Na 2  SeO 3 ) is preferred. Other selenium compounds which may be used are Na 2  SeO 4 . The total quantity of selenium added during the yeast growth in the fermenter is about 0.15 to 40% based on final dry yeast solids produced, preferably 0.2 to 0.25% and most preferred at about 0.22% selenium based on final dry yeast. 
     Additionally, the procedures of the present invention are carried out without the addition of ammonium sulfate and without the use of sulfuric acid for pH adjustment which procedures are conventional in commercial yeast manufacture processes. 
    
    
     DESCRIPTION OF THE PREFERRED EMBODIMENT 
     The following Examples 1-6 illustrate the results obtained by the use of the method of the present invention, in the propagation of selenium yeast in a ten (10) liter fermenter, as contrasted with other procedures (Examples 7) wherein a selenium salt was fed periodically to the fermenter. 
     In Examples 1-6, a calculated amount (2800 mg.) of water-soluble selenium compound, particularly sodium selenite (Na 2  SeO 3 ) was mixed with 1600 grams of molasses wort used as the carbon source nutrient added continuously over a fermentation period of 161/2 hours and was added to the yeast pitch (65 grams) in 7000 ml of water in a ten (10) liter fermenter. The nutrient medium had the following initial composition. 
     
         ______________________________________Water                 7000   ml.NH.sub.4 Cl           44     gramsNH.sub.4 H.sub.2 PO.sub.4                 16.8   gramsMgSO.sub.4.7H.sub.2 O 3.4    gramsKCl                   3.7    gramsthiamine              90     mg.Yeast                 65     grams(Saccharomyces cerevisiae)______________________________________ 
    
     Sixty milligrams of ZnSO 4 .H 2  O was added to the 1600 grams of molasses used. Ammonium hydroxide was added on demand to maintain pH at 5.2 (approximately 100 mls. of 29.8% ammonium hydroxide) throughout fermentation. Temperatures were maintained at about 29°-30° C. Aeration was continuous throughout the process to maintain aerobic yeast growth condition. 
     When the fermentation was complete, the yeast was separated, washed seven (7) times with water (1:10 dilution), reduced in moisture content to a yeast cream (16-18% yeast solids) and pasteurized at 85° C. for forty-five (45) minutes followed by drum drying to a final moisture content of about 4.5% (based on dry yeast solids). The results are shown below in Table I below. 
     
                                           TABLE I__________________________________________________________________________      Na.sub.2 SeO.sub.3 Fed with Wort (0-16.5 Hours) in 10 liter      batchesExample    1    2    3    4    5    6__________________________________________________________________________Na.sub.2 SeO.sub.3 enrichment,      2800 2800 2800 2800 2800 2800mg/fermenterSe enrichment,      1279.6           1279.6                1279.6                     1279.6                          1279.6                               1279.6mg/fermenterSe conc. in fermenter      127.96           127.96                127.96                     127.96                          127.96                               127.96beer, μg/ml (ppm)Yeast production,      420.2           420.0                414.4                     411.5                          410.8                               422.0g yeast solids/ferm.Yield % (based on      72.4 72.5 71.3 70.7 70.5 72.785° Brix molasses)__________________________________________________________________________ 
    
     EXAMPLE 7 
     In another batch yeast fermentation, the sodium selenite was added directly to the yeast fermenter in four increments at 8.5, 9.0, 9.5 and 10.0 hours of aerobic yeast growth was also made. Otherwise the fermentation procedures were the same as shown for Examples 1-6 above. Table II shows the results of this fermentation. 
     
                       TABLE II______________________________________           Na.sub.2 SeO.sub.3 Added In Four           Increments (See Above)______________________________________Na.sub.2 SeO.sub.3 enrichment,             2625(mg/fermenter)Se enrichment,    1200(mg/fermenter)Se conc. in fermenter             120beer, μg/ml (ppm)Yeast production, 260in grams of yeastsolids/fermenterYield percentage  44.2(based on 85° Brix molasses)______________________________________ [The low yield of yeast (44%) is contrasted with the higher yields (70-73%) in Examples 1-6]. 
    
     Table III is a comparison of the composition of the yeast produced from a composite of the yeasts shown in Table I (Examples 1-6) with the yeast product of Example 7 and Table II from the point of view of selenium enrichment of the yeast and the Methylene Blue Reduction Test. 
     
         ______________________________________COMPARISON OF YEAST DATAFROM TABLES I AND II        EXAMPLES 1-6 EXAMPLE 7______________________________________Se content in dried          2000           1440yeast, μg/g (ppm)Methylene Blue Reduction          90             0Test (MBRT), min.*Total Se recovered in          4997.8         386.1yeast solids, mgSe enrichment used, mg          7677.6         1200Se recovery, % 65.1           32.2N in yeast, %  9.51           4.86P.sub.2 O.sub.5 in yeast, %          3.05           3.31______________________________________ *A time of fifteen (15) minutes or greater in the MBRT test is the acceptable level for indicating that the selenium is organically bound. 
    
     From the foregoing, it is evident that high contents of organically bound intracellular selenium in yeast can be attained by feeding selenium continuously and incrementally and simultaneously with a carbon source nutrient to the yeast throughout during the yeast growth. 
     The principles of the process of the present invention may be applied to other food grade or edible yeasts other than the illustrated baker&#39;s yeast or Saccharomyces cerevisiae, such as for example, brewer&#39;s yeast or Saccharomyces uvarum or the like. While intracellular selenium contents of yeasts are preferably in the range of 1000 ppm, or more, even as high as 2500 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. 
     It should also be understood that the principles of maintaining low levels of inorganic sulfur in the nutrient media is broadly applicable to the process of producing selenium yeasts. For this reason, adjustment of pH should avoid the use of sulfuric acid as a pH regulator, in favor of non-sulfur containing pH adjustment reagents such as hydrochloric acid.