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WO2010096382A1 - Procédés de production de protéines contenant du sélénium au moyen de cellules de levure - Google Patents

Procédés de production de protéines contenant du sélénium au moyen de cellules de levure Download PDF

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WO2010096382A1
WO2010096382A1 PCT/US2010/024292 US2010024292W WO2010096382A1 WO 2010096382 A1 WO2010096382 A1 WO 2010096382A1 US 2010024292 W US2010024292 W US 2010024292W WO 2010096382 A1 WO2010096382 A1 WO 2010096382A1
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Prior art keywords
yeast
growth medium
hydroxy
acid
selenium
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Friedhelm Brinkhaus
Stephen J. Lorbert
Justin Morgenthaler
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Novus International Inc
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Novus International Inc
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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
    • C12P21/00—Preparation of peptides or proteins
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N1/00—Microorganisms, e.g. protozoa; Compositions thereof; Processes of propagating, maintaining or preserving microorganisms or compositions thereof; Processes of preparing or isolating a composition containing a microorganism; Culture media therefor
    • C12N1/14—Fungi; Culture media therefor
    • C12N1/16—Yeasts; Culture media therefor

Definitions

  • the present invention generally relates to processes for the production of proteins that include selenium using yeast cells.
  • the present invention provides processes for producing proteins that include selenium by fermenting yeast in the presence of organic selenium sources.
  • Selenium is an essential micronutrient that is critical for the biosynthesis of selenoproteins in most animals.
  • Selenium is typically incorporated into selenoproteins in the form of selenocysteine, an amino acid in which a selenium atom is substituted in place of the sulfur atom.
  • selenocysteine an amino acid in which a selenium atom is substituted in place of the sulfur atom.
  • About 30 families of selenoproteins have been identified throughout the animal kingdom, and 25 specific selenoproteins have been identified in humans. Many of these selenoproteins are redox enzymes with selenium at the active site, although the functions of some selenoproteins remain undetermined.
  • dietary selenium has anti-oxidative properties, protective effects against UV radiation, and reduces the incidence of cancers such as lung cancer, colorectal cancer and prostate cancer.
  • the organic selenium compounds produced by these organisms are typically in the form of selenomethionine that is randomly incorporated into various proteins of the organism. When ingested by animals such as mammals and birds, selenomethionine is readily converted into selenocysteine and incorporated into selenoproteins.
  • Selenomethionine may be produced in plants such as wheat, corn and soya by growing the plants in the presence of an inorganic selenium source such as sodium selenite.
  • an inorganic selenium source such as sodium selenite.
  • plant matter incorporates selenium in relatively limited amounts, and therefore must be consumed in rather sizeable servings in order to ingest an amount of selenomethionine sufficient to satisfy daily selenium requirements.
  • Yeast may also be used to produce selenomethionine.
  • Selenium yeast is a readily digestible form of selenium supplementation that may be concentrated for use as a dietary supplement or as an ingredient for vitamin enrichment of various food products. Due to the high protein content of yeast compared with plants, selenomethionine may be produced at much higher concentrations in yeast.
  • selenium yeast production overcome this limitation by adding the sodium selenite gradually over the period of active growth of the yeast, resulting in higher selenium incorporation into the yeast as selenomethionine.
  • the selenium yeast product must undergo a rinse step to eliminate any residual toxic inorganic selenium.
  • the production facility must take precautions to ensure that the selenium yeast products do not contain inorganic selenium salts, and that production workers are not exposed to toxic levels of inorganic selenium compounds.
  • a method for the production of a protein that includes selenium in a yeast cell includes fermenting yeast cells in a growth medium in the presence of an organic selenium source in a manner such that the organic selenium source is converted to a selenoamino acid residue that is incorporated into the yeast cellular protein, resulting in a protein that includes selenium.
  • a method for the production of a protein that includes selenium in a yeast cell includes fermenting the yeast cells in a growth medium in the presence of 2-hydroxy, 4- methylselenobutyhc acid in a manner such that the 2-hydroxy, 4- methylselenobutyric acid is converted to a selenomethionine residue that is incorporated into the yeast cellular protein, which now includes selenium.
  • a method for the production of a protein that includes selenium in a yeast cell in which the resulting yeast cells are essentially free of inorganic selenium compounds.
  • the method includes fermenting the yeast cells in a growth medium in the presence of 2- hydroxy, 4-methylselenobutyric acid in a manner such that the 2-hydroxy, 4- methylselenobutyric acid is converted to a selenomethionine residue that is incorporated into the yeast cellular protein comprising selenium and then purifying the yeast cells from the growth medium when the yeast are within the stationary phase of the growth curve.
  • FIG. 1 is an exemplary growth curve of yeast cells fermenting in a growth culture.
  • FIG. 2 is a growth curve of yeast cells cultured with a single dose of organic selenium source.
  • FIG. 3 is a growth curve of yeast cells cultured with an organic selenium source added to the growth medium at a steady rate.
  • FIG. 4 is a growth curve of yeast cells cultured with an organic selenium source added to the growth medium at a rate that increased as a function of the number of yeast cells in the culture.
  • Various embodiments of the present invention provide a method of producing protein that includes selenium in yeast cells.
  • the method includes fermenting the yeast cells in a growth medium that includes an organic selenium source. It has been discovered that yeast cells that are fermented in a growth medium that includes an organic selenium source such as 2-hydroxy 4- methylseleno butyric acid (HMSBA) readily incorporate selenium into the yeast proteins.
  • the resulting selenium yeast may contain at least 3000 ppm of intracellular selenium.
  • the intracellular content of the selenium yeast typically includes selenomethionine residues incorporated into the yeast's cellular proteins.
  • the yeast cells are essentially free of inorganic selenium compounds. No rinsing or washing of the yeast is necessary in any embodiment of this method to eliminate residual inorganic selenium compounds.
  • proteins that include selenium are produced by yeast cells that are fermented in a growth medium containing an organic selenium source.
  • the selenium is incorporated into the yeast as selenoamino acid residues in the proteins within the subcellular components of the yeast including but not limited to cell wall, cell membrane, mitochondria and cytosol.
  • selenoamino acid residues incorporated into the yeast cell proteins are selenomethionine and selenocysteine.
  • the yeast may also include selenium in an amount of less than about 10% of the total selenium content of the yeast in the form of other organic selenium compounds including but not limited to selenocystathione, Se-methylated selenocysteine, ⁇ -glutamyl-Se-methyl selenocysteine, ⁇ -glutamylselenomethionine, Se-adenosylselenohomocysteine, and selenolanthionine.
  • the yeast is essentially free of inorganic selenium compounds.
  • less than 1 % of the intracellular selenium content of the yeast includes inorganic selenium compounds selected from the group consisting of selenite, selenic acid, and combinations thereof.
  • At least 50% of the organic selenium source supplied to the yeast cells in the growth medium may be converted into selenoamino acid residues.
  • at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, or at least 90% of the organic selenium source supplied to the yeast cells in the growth medium may be converted into selenoamino acid residues.
  • at least 75% of the organic selenium source supplied to the yeast cells in the growth medium may be converted into selenoamino acid residues.
  • the selenium yeast resulting from an embodiment of the method may contain at least 1000 ppm of intracellular selenium.
  • the selenium yeast may contain at least 2000 ppm, at least 3000 ppm, at least 4000 ppm, at least 5000 ppm, at least 6000 ppm, at least 7000 ppm, at least 8000 ppm, at least 9000 ppm, or at least 10,000 ppm of intracellular selenium.
  • the selenium yeast may contain at least 3000 ppm of intracellular selenium.
  • the proteins that include selenium produced by the yeast cells may be provided in the form of proteins contained in intact wet yeast cells or dried yeast cells.
  • the proteins that include selenium may be extracted from intact yeast cells using known methods including but not limited to maceration, lysis, grinding, sonication, homogenization, solvent extraction, and enzymatic digestion.
  • the proteins containing selenium may be further broken down into selenoamino acid resides using known methods including but not limited to enzymatic digestion with peptidases.
  • proteins containing selenium are produced by fermenting the yeast cells in a growth medium containing an organic selenium source.
  • the yeast cells may be fermented in an aerobic fermentation vessel or an anaerobic fermentation vessel. If the yeast cells are fermented anaerobically, ethanol may be recovered from the remaining growth medium after the selenium yeast cells have been removed from the anaerobic fermentation vessel.
  • the total amount of proteins containing selenium produced by the yeast cells is governed by at least two factors: the total selenium content of each yeast cell and the total number of yeast cells produced.
  • the amount of selenium incorporated into the yeast proteins as selenoamino acid residues is proportional to the concentration of organic selenium source in the growth medium up to a saturation level.
  • elevated levels of organic selenium source in the growth medium may also inhibit the reproduction of the yeast cells, resulting in fewer yeast cells, as illustrated in the examples below.
  • the amount of organic selenium source included in the growth medium may be selected to balance these two factors.
  • the selenium content of each yeast cell may be sensitive to the timing of the addition of the organic selenium source to the growth medium.
  • yeast growth in culture follows a stereotypical growth curve, illustrated in FIG. 1.
  • the yeast cells are biochemically active, but not actively dividing; the number of yeast cells remains at a relatively constant initial level during a subsequent lag phase.
  • the yeast cells actively metabolize and reproduce, causing a rapid increase in the number of yeast cells.
  • the metabolism of the yeast cells slow, rapid cell division ceases, and the number of yeast cells remains at a high final level.
  • the organic selenium source may be added to the growth medium at any given time.
  • the organic selenium source may be added to the growth medium in a single dose at a selected time during the exponential growth phase of the yeast.
  • the organic selenium source may be added to the growth medium in at least two or more discrete doses at selected times during the exponential growth phase of the yeast.
  • the organic selenium source may be added to the growth medium continuously during the exponential growth phase of the yeast.
  • the initial time at which the organic selenium source is added to the growth medium may be timed to coincide with the initiation of the exponential growth phase of the yeast cells.
  • the time at which the exponential growth phase commences may be estimated using factors including but not limited to previously measured growth curves, the composition and temperature of the growth medium, and the species of yeast in the growth medium.
  • the organic selenium source may be added after the yeast has fermented in the growth medium for a period of between about 12 hours and about 24 hours when the yeast species is Saccharomyces cerevisiae.
  • the organic selenium source may be added after the yeast has fermented in the growth medium for a period of between about 8 hours and about 24 hours, between about 10 hours and about 20 hours, between about 10 hours and about 16 hours, or between about 11 hours and about 13 hours. In an exemplary embodiment, the organic selenium source may be added after about 12 hours of fermentation when the yeast species is Saccharomyces cerevisiae.
  • the time at which the exponential growth phase commences may be determined by monitoring changes in the characteristics of the yeast cells.
  • characteristics of the yeast cells include cell density, number of cells, cell morphology, and cell metabolism.
  • the number of cells may be monitored using known devices including but not limited to haemocytometers, Coulter counters, or laser-flow cytometers, scales may be used measure the wet or dry weight of the cells. Centrifuges may be used to determine cell volume, and spectrophotometers may be used to measure the optical density of the cells.
  • Cell morphology may be monitored by microscopic image analysis.
  • the organic selenium source may be added to the yeast growth medium when the average mass of the yeast cells exceeds at least 35 g/L. In other embodiments, the organic selenium source may be added to the yeast growth medium when the average mass of the yeast cells exceeds at least 35 g/L, at least 40 g/L, at least 50 g/L, at least 60 g/L, at least 70 g/L, or at least 80 g/L.
  • the organic selenium source may be introduced at a rate of about 50 mg/hr/kg of yeast cells. In other embodiments, the organic selenium source may be introduced at a rate of about 45 mg/hr/kg of yeast cells, about 40 mg/hr/kg of yeast cells, about 30 mg/hr/kg of yeast cells, about 20 mg/hr/kg of yeast cells, or about 10 mg/hr/kg of yeast cells, starting at an initial rate of about 5 mg/hr during the experimental phase of the growth area.
  • the yeast is grown under aerobic conditions and the organic selenium source may be added to the growth medium after the dissolved oxygen concentration of the growth medium has decreased below about 90% of the initial dissolved oxygen concentration.
  • the yeast may be grown under aerobic conditions and the organic selenium source may be added to the growth medium after the dissolved oxygen concentration has decreased below about 90%, below about 85%, below about 80%, below about 75%, below about 50%, or below about 25% of the initial dissolved oxygen concentration.
  • the organic selenium source may be added continuously at a steady and constant rate, or alternatively at a rate that varies with time according to a predetermined schedule.
  • the organic selenium source may be added continuously at a rate that varies as a function of measured characteristics of the yeast cells in the growth medium.
  • yeast cell characteristics that may be monitored to regulate the rate of addition of organic selenium source include oxygen consumption, carbon dioxide production, heat production, ethanol production, density of cells in the growth medium, cell mass, rate of increase of cells, optical density, selenium uptake rate, NADH fluorescence, and glucose uptake rate.
  • the precise amount of organic selenium source added to the growth medium can and will vary without departing from the scope of the invention.
  • the organic selenium source may be added to the growth medium up to a concentration of about 2 mg/ml.
  • the amount of organic selenium source added to the growth medium results in a concentration of organic selenium source of up to about 1 mg/ml.
  • the rate at which the organic selenium source is added to the growth medium also can and will vary.
  • the organic selenium source may be added to the growth medium at a constant rate of less than about 6 mg/L/hr when the yeast strain is Saccharomyces cerevisiae and the organic selenium source is HMSBA.
  • the organic selenium source may be added to the growth medium at a constant rate of less than about 5 mg/L/hr, less than about 4 mg/L/hr, less than about 3 mg/L/hr, less than about 2 mg/L/hr, less than about 1 mg/L/hr, or less than about 0.5 mg/L/hr.
  • the organic selenium source included in the growth medium may be any organic compound containing at least one selenium atom in its molecule.
  • the organic selenium source may be a selenohydroxy acid compound having the formula (I):
  • R 2 is selected from ⁇ - ⁇ OH, ⁇ - ⁇ R 3 , and ⁇ - ⁇ NHR 7 ;
  • R 3 is selected from alkoxyl (Ci-C 2 6), ceramide 1 , ceramide 2, ceramide 3, ceramide 4, ceramide 5, ceramide 6a or 6b, S-cysteinyl, or S- glutathionyl, carnitoyl, lipids, polyols;
  • OR 4 is selected from alkoxyl (C1-C26), ceramide 1 , ceramide 2, ceramide 3, ceramide 4, ceramide 5, ceramide 6a or 6b, S-cysteinyl, or S-glutathionyl, carnitoyl, lipids, polyols;
  • OR 5 is selected from alkoxyl (C1-C26), ceramide 1 , ceramide 2, ceramide 3,
  • the selenohydroxy acid compound may have the formula (Ia):
  • R 1 is selected from the group consisting of hydroxyl, amino, ⁇ - ⁇ OCOR 2 , and ⁇ - ⁇ NHCOR 2 ;
  • R 2 is an organic acid derivative; and
  • n is an integer from 1 to 3.
  • Non-limiting examples of selenohydroxy acid compounds include 2-hydroxy-4-methylselenobutyric acid (HMSBA), L-2-hydroxy-4- methylselenobutyric acid, D-2-hydroxy-4-methylselenobutyric acid, D,L-2- hydroxy-4-methylselenobutyric acid, 2-ceto-4-methylselenobutyric acid, dicyclohexylammonium L-2-hydroxy-4-methylseleno-butyrate, sodium D,L-2- hydroxy-4-methylseleno-butyrate, calcium D,L-2-hydroxy-4-methylseleno- butyrate, ethyl-D,L-2-hydroxy-4-methylselenobutyrate, isopropyl-D,L-2-hydroxy- 4-methylselenobutyrate, D,L-2-acetoxy-4-methylselenobutyhc acid, D,L-2- linoleyloxy-4-methylselenobuty
  • the organic selenium source may be dissolved in an aqueous solution with an additional carbon source, described below, so long as the concentration of the organic selenium source falls within the limits described above.
  • the yeast used to produce proteins containing selenium may be any food grade or edible yeast.
  • Non-limiting examples of food-grade or edible yeasts include Saccharomyces cerevisae, Saccharomyces boulardii, Saccharomyces torula, Saccharomyces exiguous, Candida stellata, Schizosaccharomyces pombe, Torulaspora delbrueckii, and Zygosaccharomyces bailii.
  • the yeast may be a species typically used for the brewing of fermented beverages, including but not limited to Brettanomyces lambicus, Brettanomyces bruxellensis, Brettanomyces claussenii, and Saccharomyces pastorianus.
  • the yeast may be genetically engineered strains typically used for large- scale ethanol production including but not limited to various engineered strains of Saccharomyces species.
  • the yeast Before adding the yeast to the growth medium, the yeast may be streaked onto agar plates and cultivated at a temperature of about 30° C for a period ranging between about 48 and 96 hours, to cultivate a sterile yeast colony.
  • a yeast inoculum may be formed by scraping an amount of yeast from the agar plate into a sterile inoculum medium.
  • the inoculum may be added to the growth medium in the fermentation vessel immediately after adding the yeast cells, or the inoculum may be cultivated for a period of up to about 72 hours to increase the number of yeast cells in the inoculum prior to use.
  • the inoculum medium refers to an aqueous solution containing the nutrients necessary for yeast growth.
  • the inoculum medium may include a source of carbon such as molasses, simple sugars such as glucose, sucrose, dextrose, fructose, and maltose, and combinations thereof.
  • the inoculum medium may also include a source of amino acids and nitrogen such as yeast extract, bacto-peptone, ammonia, ammonium dihydrogen phosphate, and combinations thereof.
  • the inoculum medium may be an aqueous solution comprising yeast extract at a concentration ranging from about 5 g/L to about 20 g/L, bacto-peptone at a concentration ranging from about 10 g/L to about 30 g/L, and glucose at a concentration ranging from about 10 g/L to about 30 g/L.
  • the growth medium refers to any aqueous solution containing the nutrients necessary for yeast growth.
  • the growth medium may include carbon sources including but not limited to: molasses, simple sugars including glucose, sucrose, dextrose, fructose, and maltose, and combinations thereof.
  • the growth medium may also include a source of amino acids and nitrogen including but not limited to: yeast extract, bacto-peptone, ammonia, ammonium dihydrogen phosphate, and combinations thereof.
  • the growth medium may further include other nutrient salts to supply the growing yeast with other vitamins and minerals.
  • Non-limiting examples of other nutrient salts include biotin, vitamin Bi, vitamin B 6 , calcium pantothenoate, calcium sulfate, inositol, copper, copper sulfate, zinc, zinc sulfate, iron, iron sulfate, potassium, potassium sulfate, magnesium, magnesium sulfate, potassium hydroxide, potassium sulfate, phosphoric acid, sodium iodide, sodium molybdate, boric acid, cobalt chloride, zinc chloride, sulfuric acid, phosphoric acid, and combinations thereof.
  • the growth medium may be an aqueous solution that includes calcium sulfate at a concentration ranging from about 0.8 g/L to about 1.1 g/L, potassium sulfate at a concentration ranging from about 10 g/L to about 30 g/L, magnesium sulfate at a concentration ranging from about 5 g/L to about 25 g/L, potassium hydroxide at a concentration ranging from about 2 g/L to about 8 g/L, phosphoric acid at a concentration ranging from about 10 g/L to about 40 g/L, yeast extract at a concentration ranging between about 5 g/L and about 15 g/L, glucose at a concentration ranging between about 40 g/L and about 60 g/L, an anti-foam composition at a concentration ranging between about 5 g/L and about 10 ml/L and a mixture of trace metals at a concentration ranging from about 2 ml/L to about 8
  • the mixture of trace metals may be an aqueous solution that includes copper sulfate in an amount ranging between about 0.5% and about 0.6% by weight, sodium iodide in an amount ranging between about 0.005% and about 0.1 % by weight, manganese sulfate in an amount ranging between about 0.1 % and about 0.3% by weight, sodium molybdate in an amount ranging between about 0.01 % and about 0.03% by weight, boric acid in an amount ranging between about 0.001 % and 0.003% by weight, cobalt chloride in an amount ranging between about 0.03% and about 0.05% by weight, zinc chloride in an amount ranging between about 1 % and about 3% by weight, iron sulfate in an amount ranging between about 5% and about 7% by weight, biotin in an amount ranging between about 0.01 % and about 0.03% by weight, and sulfuric acid in an amount ranging between about 0.3% and about 0.5% by weight.
  • additional amounts of a carbon source may be added to the growth medium.
  • an additional amount of carbon source may be added to the growth medium continuously, in discrete boluses at selected times, or in a single dose at a selected time during the production of the selenium yeast.
  • the additional carbon source may be an aqueous solution having a concentration ranging from about 5% to about 70% by weight.
  • the carbon source may be a 17% glucose solution by weight and may be added continuously to growth medium after the yeast has fermented for about 12 hours, when the yeast species is Saccharomyces cerevisiae.
  • the rate at which a carbon source is added to the fermentation vessel may vary depending on some characteristic of the yeast or growth medium in the fermentation vessel.
  • the characteristics of the yeast or growth medium include dissolved oxygen concentration, yeast cell density, ethanol concentration, pH, temperature, carbon dioxide concentration, and combinations thereof.
  • a fermentation vessel as defined herein refers to any sterile container capable of holding the yeast and growth medium while the yeast is fermenting.
  • the fermentation vessel may be capable of conducting aerobic fermentation or anaerobic fermentation.
  • Non-limiting examples of fermentation vessels known in the art include beakers, flasks, shake flasks, tanks, vats, fermentors, incubators, and bioreactors.
  • the fermentation vessel may be capable of actively monitoring and adjusting various aspects of the fermentation environment including but not limited to: agitation of the growth medium; dissolved gas concentration within the growth medium; temperature of the growth medium; pressure of the growth medium; pH of the growth medium; nutrient concentrations in the growth medium; density of yeast cells in the growth medium, and combinations thereof.
  • the fermentation vessel may be a commercially available fermentor with adjustable agitation speed, adjustable rate of flow of a mixture of gases including but not limited to nitrogen, oxygen and carbon dioxide through the growth medium, and controllable rate of addition of nutrients.
  • a mixture of gases comprising nitrogen, carbon dioxide, and combinations thereof may flow through the growth culture to exclude oxygen from the growth culture.
  • air or pure oxygen may flow through the growth medium to induce aerobic metabolism by the fermenting yeast cells.
  • the fermentation vessel may be a commercially available fermentor having an agitation speed ranging from about 600 to 1000 rpm, continuous introduction of air through the growth medium at a rate of about one vessel volume per minute, active control of the pH of the growth medium to a desired value by adding an acid or base; and the introduction of nutrients in aqueous solution to the growth medium at a rate controlled by the level of dissolved oxygen levels in the growth medium.
  • a 17% solution of glucose by weight may be added to the fermentation vessel at a rate sufficient to limit the rate of growth of the yeast colony to a level that sustains a dissolved oxygen concentration of at least 90% of its initial level in the growth medium.
  • An aqueous solution containing the organic selenium source may be added to the fermentation vessel at a controlled rate of addition defined above.
  • the yeast cells containing proteins that include selenium may be purified from the growth medium after the yeast growth has reached a predetermined level. Although the selenium yeast cells may be recovered at any time after the organic selenium source is added to the growth medium, the selenium yeast cells are typically recovered at a time at which the maximum number of yeast cells is achieved. The yeast may be purified when the yeast is within the stationary phase of the growth curve as previously described and illustrated in FIG. 1.
  • the time at which the yeast cells enter the stationary phase may be estimated based on previously measured growth curves for yeast cultures having comparable fermentation conditions.
  • the yeast cells may be recovered from the growth medium after about 40 hours of fermentation when the yeast strain is Saccharomyces cerevisiae and the organic selenium source is HMSBA.
  • the time at which the yeast cells enter the stationary phase may be determined by monitoring the stabilization of various characteristics of the morphology or metabolism of the yeast cells in the growth media.
  • these various characteristics include: number of cells, density of cells, mass of cells, optical density of cells; temperature; pH; concentration of glucose or other carbon sources; dissolved oxygen concentration; concentration of carbon dioxide; fluorescence of NADH, viscosity; and redox potential.
  • the yeast cells may be purified from the growth medium when the level of yeast growth ranges from about 100 g of cells/L of growth medium to about 500 g of cells/L of growth medium.
  • the yeast may be purified from the growth medium when the level of yeast growth ranges from about 100 g of cells/L to about 200 g of cells /L, from about 150 g of cells/L to about 250 g of cells /L, from about 200 g of cells/L to about 300 g of cells /L, from about 250 g of cells of cells /L to about 350 g of cells /L, from about 300 g of cells/L to about 400 g of cells /L, from about 350 g of cells/L to about 450 g of cells /L, or from about 400 g of cells/L to about 500 g of cells /L of growth medium.
  • the yeast cells may be purified from the growth medium using methods known in the art.
  • methods suitable for purifying the yeast include centhfuging the yeast cells out of the growth medium and filtering the yeast cells out of the growth medium.
  • the purified yeast cells may be used without need for further rinsing or cleansing.
  • the purified yeast cells may be additionally pasteurized and dried after they are purified from the growth medium.
  • the pasteurization may be performed by heating the purified yeast cells to a temperature ranging between about 30 0 C and about 110 0 C.
  • the purified cells may be further dried using various methods known in the art, including but not limited to freeze-drying, drum drying, tray drying, and spray drying.
  • the protein that includes selenium may be purified from the yeast cells using known methods such as maceration or homogenization, as described above.
  • the selenoamino acid residues including but not limited to selenomethionine and selenocysteine may be purified from the proteins containing selenium by known methods including but not limited to enzymatic digestion using a peptidase, as described above.
  • the remaining growth medium may contain ethanol after the yeast cells have been purified.
  • the ethanol may be recovered from the growth medium by any appropriate method known in the art. Non-limiting examples of ethanol recovery methods include fractional distillation, molecular sieving, pressure reduction, and membrane filtration.
  • the proteins containing selenium resulting from the method of the present invention may be used for a variety of nutritional applications.
  • Wet or dried intact yeast cells that include proteins containing selenium may be used as an additive for selenium-enriched food products, as an ingredient in dietary supplement formulations, or as a nutritional additive to the feeds of livestock and other domestic animals.
  • Purified proteins containing selenium or purified selenoamino acids may be used as ingredients in pharmaceutical compositions or as ingredients in cosmetic formulations, in addition to the nutritional uses described above for intact yeast cells.
  • EXAMPLE 1 Selenium yeast cells were produced under aerobic conditions by introducing HMSBA in a single dose into the growth media.
  • Saccharomyces yeast was streaked onto agar plates and cultivated at a temperature of about 30° C for about 72 hours.
  • An inoculum was prepared by scraping the yeast off of the plate into 50 ml of YPD medium containing 10 g/L of yeast extract, 20g/L of bacto-peptone, and 20g/L of glucose, and 4.4 ml/L of a trace metals solution.
  • the trace metals solution included 6 g/L of cupric sulfate, 0.08 g/L of sodium iodide, 3 g/L of manganese sulfate, 0.2 g/L of sodium molybdate, 0.02 g/L of boric acid, 0.5 g/L of cobalt chloride, 20 g/L of zinc chloride, 65 g/L of ferrous sulfate, 0.2g/L of biotin, and 0.5% by volume sulfuric acid.
  • the inoculum was allowed to grow in the YPD medium for several hours.
  • HMSBA 0.95 L of the YPD growth medium described above.
  • HMSBA was added to the nutrient mixture in a single dose of 34.1 g, 162.7g, 258g, and 524g. All shake flasks were placed in an incubator shaker at a temperature of 30° C and incubated for at least 72 hours.
  • the total organic selenium concentration of the yeast increased when higher amounts of HMSBA were added to the growth media.
  • the HMSBA also inhibited the growth of the yeast cells, as shown in FIG. 2, resulting in less yeast biomass in the cultures with higher amounts of added HMSBA.
  • the conversion rate defined as the percent of HMSBA added to the growth medium that was converted into organic selenium compounds by the yeast, was lowest for the cultures with the highest amount of HMSBA added.
  • HMSBA was converted into organic selenium compounds in the yeast cells when added to the growth medium of the yeast.
  • HMSBA also inhibits the growth of yeast at higher concentrations
  • the introduction of HMSBA as a selenium source for the cultivation of selenium yeast must provide an amount of HMSBA to produce sufficient concentrations of organic selenium compounds in the yeast, while avoiding the inhibitory effect of HMSBA on yeast growth at high HMSBA concentrations.
  • EXAMPLE 2 Selenium yeast cells were produced under anaerobic conditions by introducing HMSBA in a single dose into the growth media.
  • Saccharomyces spp. yeast inoculum was prepared using the methods described in Example 1. The inoculum was added to shake flasks containing 0.95 L of growth medium as described in Example 1. HMSBA was added to the nutrient mixture in a single dose in an amount of Og, 5Og, and 100g. The air inside all shake flasks was replaced by nitrogen, and the flasks were sealed and placed in an incubator shaker at a temperature of 30° C and incubated for at least 72 hours.
  • HMSBA may be used as a selenium source for producing selenium yeast under anaerobic conditions, but that HMSBA also exerted a dose-dependent inhibitory effect on yeast cell growth.
  • EXAMPLE 3 Selenium yeast cells were produced under aerobic conditions by introducing HMSBA continuously into the growth media.
  • Each inoculum was added to the fermentation vessel of a Bioflo 110 fermentor (New Brunswick Scientific, Edison, NJ, USA) containing 0.95 L of growth medium that included 0.94g/L of calcium sulfate, 18.2 g/L of KSO 4 , 15 g/L of MgSO 4 , 4.0 g/L of KOH, 26 g/L of phosphoric acid, 10 g/L of yeast extract, 7.0 mL/L of an anti-foam formulation, 50 g/L of glucose, and 4.4 ml/L of the trace metal composition previously described in Example 1. Airflow was bubbled through the growth medium at a rate of 1 vessel volume per minute and agitation of the growth medium was initiated at 600 rpm.
  • Dissolved oxygen in the growth medium was monitored during fermentation and the agitation was increased up to a maximum of 1000 rpm to maintain the dissolved oxygen level at 95% of the initial level for as long as possible.
  • Glucose (170g/L solution in water) was added to the growth medium at a controlled rate to limit the rate of growth such that the dissolved oxygen concentration was maintained above 90% of the initial value for as long as possible.
  • FIG. 4 shows a similar growth curve for a yeast culture to which a total of 1200 mg of HMSBA was added to the nutrient medium, at an HMSBA concentration of 1.2mg/ml.

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Abstract

La présente invention concerne un procédé de production d'une protéine contenant du sélénium par fermentation de levures dans un milieu de culture comprenant une source de sélénium organique.
PCT/US2010/024292 2009-02-17 2010-02-16 Procédés de production de protéines contenant du sélénium au moyen de cellules de levure Ceased WO2010096382A1 (fr)

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CN105441487A (zh) * 2014-09-03 2016-03-30 赵小勇 生物有机硒及其制备方法和用途及由其制备的饲料或肥料
CN108794566A (zh) * 2018-06-29 2018-11-13 张新江 一种复合酶二步酶解高硒西兰花及磨浆法制备硒蛋白的方法
CN112251365A (zh) * 2020-10-16 2021-01-22 大连澎立生物科技有限公司 酵母蛋白硒及其制备方法、发酵培养基、富硒小分子肽原液及其制备方法和食品
JP2021521839A (ja) * 2018-04-27 2021-08-30 ルサッフル・エ・コンパニーＬｅｓａｆｆｒｅ Ｅｔ Ｃｏｍｐａｇｎｉｅ 酵母タンパク質
WO2023010910A1 (fr) * 2021-08-04 2023-02-09 安琪酵母股份有限公司 Levure enrichie en sélénium organique, son procédé de préparation, produit et application
CN116144513A (zh) * 2022-12-26 2023-05-23 自然资源部第三海洋研究所 一种具富硒及抑菌功能的发酵性拉茜斯酵母及其应用
CN119530037A (zh) * 2025-01-22 2025-02-28 江苏惠利生物科技有限公司 一种有机硒培养基及其在制备富硒酵母中的应用

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Cited By (11)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN105441487A (zh) * 2014-09-03 2016-03-30 赵小勇 生物有机硒及其制备方法和用途及由其制备的饲料或肥料
JP2021521839A (ja) * 2018-04-27 2021-08-30 ルサッフル・エ・コンパニーＬｅｓａｆｆｒｅ Ｅｔ Ｃｏｍｐａｇｎｉｅ 酵母タンパク質
US11602156B2 (en) 2018-04-27 2023-03-14 Lesaffre Et Compagnie Yeast proteins
JP7433243B2 (ja) 2018-04-27 2024-02-19 ルサッフル・エ・コンパニー 酵母タンパク質
US11937620B2 (en) 2018-04-27 2024-03-26 Lesaffre Et Compagnie Yeast proteins
CN108794566A (zh) * 2018-06-29 2018-11-13 张新江 一种复合酶二步酶解高硒西兰花及磨浆法制备硒蛋白的方法
CN108794566B (zh) * 2018-06-29 2021-12-03 恩施硒圣植物科技有限公司 一种复合酶二步酶解高硒西兰花制备硒蛋白的方法
CN112251365A (zh) * 2020-10-16 2021-01-22 大连澎立生物科技有限公司 酵母蛋白硒及其制备方法、发酵培养基、富硒小分子肽原液及其制备方法和食品
WO2023010910A1 (fr) * 2021-08-04 2023-02-09 安琪酵母股份有限公司 Levure enrichie en sélénium organique, son procédé de préparation, produit et application
CN116144513A (zh) * 2022-12-26 2023-05-23 自然资源部第三海洋研究所 一种具富硒及抑菌功能的发酵性拉茜斯酵母及其应用
CN119530037A (zh) * 2025-01-22 2025-02-28 江苏惠利生物科技有限公司 一种有机硒培养基及其在制备富硒酵母中的应用

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