Nitrogen nutri+on and assimila+on in Saccharomyces fermenta+ons. William Pengelly, Ph.D. MBAA District NW Hood River, OR June 1-2, 2012
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1 Nitrogen nutri+on and assimila+on in Saccharomyces fermenta+ons William Pengelly, Ph.D. MBAA District NW Hood River, OR June 1-2, 2012
2 Importance of Nitrogen Nitrogen is a major component of nearly all complex biomolecules important in the structure and func+on of living organisms.
3 Nitrogen in biomolecules 1. Amino Acids Pep+des and Proteins. 2. Purines, Pyrimidines Nucleo+des (ATP, GTP), Nucleic Acids (DNA, RNA) 3. Vitamins, Enzyme Cofactors 4. Lipids: Phospha+dyl serine, phospha+dyl ethanolamine, lecithin. 5. Polyamines (DNA func+on) 6. Chi+n (cell walls)
4 Source of Nitrogen for important Vitamins and Cofactors VITAMIN COENZYME FUNCTION SOURCE OF N IN BIOSYNTHESIS Biotin Carboxylation Alanine, Methionine Cobalamine (B12) Alkylation Glycine Folic Acid Single Carbon Group Transfer Glutamine, Aspartate, Glycine Nicotinamide (B3) Oxidation-Reduction Aspartate Pantothenic Acid (B5) (CoA) Acyl Transfer Methionine Pyridoxine (B6) Amino Group Transfer Threonine Riboflavin (B2) Oxidation-Reduction Glutamine, Aspartate, Glycine Thiamine (B1) Aldehyde Transfer Tyrosine, Cysteine
5 Sources of Nitrogen 1. N 2 : 80% of atmosphere. Nitrogen fixa+on by cyanobacteria, legume/ Rhizobium symbiosis, or woody plant/ Frankia symbiosis. 2. NO 3 : Nitrate reduc+on by plants and microbes. 3. Brewer s Yeast requires reduced forms of nitrogen: amino acids, urea, uric acid, ammonia, etc.
6 AMMONIA ASSIMILATION IN YEAST
7 AMMONIA ASSIMILATION IN YEAST
8 ARE AMINO ACIDS ASSIMILATED AS IS? 1. The complement of amino acids in barley storage proteins is not the same as that needed by yeast for growth. 2. Amino acids are absorbed by yeast differenfally: Group A: Absorbed immediately during first 20 hr. Group B: Absorbed gradually during fermentafon. Group C: Absorbed amer an a lag. Group D: Not aborbed. 3. Margaret Jones, Michael J Pragnell, and John S. Pierce, AbsorpFon of Amino Acids by Yeasts from a Semi- Defined Medium SimulaFng Wort. J. Inst. Brewing 75: (1975)
9 Jones et al. (1975) J. Inst. Brewing 75: C and 15 N labeled amino acids. Automated amino acid analysis: Column chromatography separa+on, post column ninhydrin reac+on, spectrophotometric quan+ta+on. Split column for isotope detec+on.
10 Jones et al. (1975) J. Inst. Brewing 75: C carbon skeletons can find their way into protein. 14 C carbon skeletons are recycled. 15 N indicate random distribu+on of 15 N throughout all amino acids. Glutamic Acid is at the center of all transamina+on systems within the yeast cell.
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12 α-keto acid 2 + amino acid 1 = α-keto acid 1 + amino acid 2
13 The Ehrlich Pathway
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15 Thermodynamics of Transamina+on (Hazelwood et al. (2008) Appl. Env. Microbiol. 74: ) Phenylalanine + α- Ketoglutarate Phenylpyruvic acid + Glutamate (52 μ mole/l) (36 μ mole/l) (5,850 μmole/l) Ehrlich Pathway (Wu et al Metabol. Eng. 8: )
16 Insufficient Nitrogen Nutri+on Importance of C/N ra+o. Adequate N in North American 2- row for all- malt beers; Total Protein = 11%, S/T = 0.44, FAN = 200. What is the op+mal C/N ra+o? Gibson, Brian R. (2011) 125 th Anniversary Review: Improvement of Higher Gravity Brewery Fermenta+on via Wort Enrichment and Supplementa+on. J. Inst. Brewing 117:
17 Common Adjuncts and Fermentables Sugar (zero nitrogen) Unmalted grain (wheat, barley, rice, corn, etc.) Flaked products (wheat, barley, rice, corn, rolled oats, etc). Fruit (Fruit wines generally require nitrogen supplements) Honey ( % amino acids) Gluten- free. Millet (11%), Rice (7-8%), Sorghum (11-13%), Buckwheat (13-15%). Don t worry about caramel or dark roasted malts.
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22 Insufficient N: What happens? Ulrik Schulze, Gunnar Liden, Jens Nielsen, and John Villadsen (1996) Physiological effects of nitrogen starva+on in an anaerobic batch culture of Saccharomyces cerevisiae, Microbiology 142: g/L and NH g/L.
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26 Flavor: Ammonia vs. Amino Acids 1. Fewer fusel alcohols with ammonia. 2. More glycerol with ammonia. 3. Esters? Acetate esters, including ethyl acetate, promoted by nitrogen rich media. Both ammonia and amino acids can induce alcohol acetyl transferases. 4. Slightly lower ethanol with ammonia. 5. Diacetyl produc+on expected to be higher with ammonia. 6. Direct flavor impact of amino acids: Proline
27 PROLINE
28 Gracias y Salud!
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SPECIMEN VALIDITY per creatinine INTERVAL 2.5 th 16 th 50 th 84 th 97.5 th Creatinine 57 mg/dl 15-120 Glutamine/Glutamate 4.3 5-160 Ammonia Level (NH 4 ) 42800 μm/g 18000-100000 Specimen Validity Index
INGREDIENT STATEMENTS FOR ES FOODS BREAKFAST BREAKS 06/29/12
INGREDIENT STATEMENTS FOR ES FOODS BREAKFAST BREAKS 06/29/12 ESF 61101 Breakfast Breaks Cocoa Puffs Reduced Sugar: Cocoa Puffs Reduced Sugar: Whole Grain Corn, Sugar, Corn Meal, Corn Syrup, Cocoa Processed