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Maintaining Ideal Yeast Health: Nutrients Yeast Need Eric Molson “What makes a great beer? Fermentation. You put the yeast in, and then the fermentation takes about a week. A lot happens in that week. The trick is to give the yeast exactly what it wants. It’s a mould and it wants to eat, and while it eats, it makes alcohol and gas.” Interview with Eric Molson; Globe and Mail, May 29, 2009 General concept Yeast Wort nutrients Beer Importance of Yeast to Beer Characteristics • Around 950 compounds are thought to give beer its character • Of these, around 400 are produced by yeast! WORT (sugars, amino acids, etc.) Yeast autolytic compounds More yeast! Acids (organic & fatty) Phenols Alcohols (ethanol & fusel oils) Sulphur compounds CO2 Aldehydes & ketones Glycerol Esters Elemental requirements for growth of brewing yeast Macroelements H - cell water, organic material O - respiration, cell water, organics C - organic material N - proteins, nucleic acids, coenzymes S - proteins, coenzymes P - nucleic acids, phosphoslipids K - cell electrolyte, enzyme cofactor Mg - enzyme cofactor, cell division Microelements Ca - flocculation Mn - enzyme cofactor Fe - cytochromes Zn - alcohol dehydrogenase Co - vitamin B12, coenzymes Cu, Ni, Mo - specialised enzymes Yeast nutrients in wort • Carbohydrates (fermentable sugars) • Nitrogen sources (mainly amino acids) • Inorganic sources (P, S, Mg, Zn, other minerals) • Oxygen (need around 25% saturation of wort) • Yeast “foods” (vitamins, fatty acids, sterols) NOTE: The level and availability of such nutrients is very important in governing fermentation performance of brewing yeast Wort composition influences: • Rate of attenuation • Degree of attenuation • Amount of yeast produced • Flocculation • Beer characteristics Carbon sources for brewing yeast • Hexose sugars: glucose, fructose • Pentose sugars: no (but possibly xylulose) • Disaccharides: maltose, sucrose, melibiose • Trisaccahrides: maltotriose (slowly) • Tetrasaccharides: maltotetraose (possibly some strains) • Oligosaccharides: maltodextrins (unfermented) • Polysaccharides: starch (unfermented) • Others: ethanol, acetate, glycerol (only respired) Nitrogen sources for brewing yeast • Peptides • Amino acids • Ammonium salts (little present in wort) Inorganic sources for brewing yeasts Phosphorus, sulphur and potassium generally sufficient in wort Metal cations may occasionally be deficient (and ratios of metal ions may be sub-optimal) Essential bulk ions: K, Mg Essential trace ions: Zn, Ca, Fe, Mn Toxic trace ions: Pb, Cd, Cr, Hg, Cu etc. Zinc • Faster fermentation • Better flocculation • Stimulates uptake of maltose and maltotriose • Stimulation of protein synthesis and yeast growth • Protection of enzymes • Stabilisation of protein and membrane system Zinc Concentration in Wort • During wort preparation approx. 95 % of zinc from malt are lost with spent grain • In modern stainless steel breweries the zinc concentration is not sufficient (<0.2 mg/L) Slow or stuck fermentation Lager brewing yeast strain – lab conditions 100 0.6 90 0.54 80 0.48 70 0.42 60 0.36 50 0.3 40 0.24 30 0.18 20 0.12 10 0.06 LAGER strain-25°C 100 90 80 0 Percentage Zn cell associated (%) 0 Zn supernatant (ppml) Zn cell associated (fg/cell) LAGER strain-25°C 0 2 4 6 8 10 12 14 16 18 20 22 24 Tim e (h) Cell Supernatant • Zinc wort content is almost zero after 1 hour • Zn is entirely accumulated into cells which redistribute the metal among growing yeast 70 60 50 40 30 20 10 0 0 1 2 3 4 5 6 7 Tim e (h) G. Walker, ASBC 2007, Victoria, Canada Zn & fermentation performance Specific gravity • Low zinc (0.05 ppm) Specific gravity 1.060 1.050 delayed sugar utilization 1.040 1.030 1.020 1.010 1.000 0 48 96 144 192 264 Time (hours) 0 0.05 0.12 0.48 1.07 10.8 (Zn ppm) Ethanol 7 • High zinc (10 ppm) slightly delayed sugar utilization – but same after 14 days fermentation Ethanol (%) 6 5 • 0.4 ppm-1.0 ppm Zn resulted in fastest fermentation rates 4 3 2 1 0 48 96 0 144 Time (hours) 0.05 0.12 0.48 1.07 192 264 10.83 (Zn ppm) G. Walker, ASBC 2007, Victoria, Canada Magnesium • Absolutely essential (growth, fermentation) • Cell Mg correlates with viability/vitality • Required for glycolysis (stimulates fermentation) • Maintains structural integrity of yeast (stressprotectant) • Ca/Mg ratio is important – (a major consideration for commercial yeast food formulations) Vitamin sources for brewing yeasts Wort generally sufficient in folic acid, thiamine, riboflavin, pantothenic acid, niacin, inositol and biotin Biotin, however, may occasionally be deficient Can supplement wort with “yeast foods” Typical levels of vitamins in wort VITAMIN LEVEL IN WORT /100ml METABOLIC ROLES Biotin 0.5µg Carboxylations Thiamin (B1) 60µg Decarboxylations Pantothenate (B5) ~50µg Acetylations, CoA Nicotinic acid (B3) 1000µg Coenzymes Riboflavin (B2) 50µg Pyridoxine (B6) 85µg Coenzymes Amino acid metabolism Inositol 10mg Membranes Yeast transport strategies for wort nutrients WORT NUTRIENTS Yeast cell YEAST METABOLITES (beer) ? ? Translocation of wort nutrients into brewing yeast CELLULAR BARRIERS capsule, cell wall, periplasm, plasma membrane, organelles PHYSICO-CHEMICAL BARRIERS chelation, adsorption, molecular size, binding (e.g. nutrient unavailability in wort particles) PHYSICO-CHEMICAL BARRIERS • Zinc chelates with phytic acid, proteins & polyphenols • These complex compounds are very stable • Only ionic zinc is available to yeast • Regular AAS is a destructive method, which measures total zinc not available zinc • Ion-exchange separation separates complex and ionic zinc before AAS Total and complex zinc concentration of industrial wort samples 0.12 total zinc complex zinc available zinc zinc concentration [mg/L] 0.1 0.08 0.06 0.04 0.02 0 1 2 3 4 5 6 Derived from: Vecseri-Hegyes, B., P. Fodor, et al. (2005). "The Role of Zinc in Beer Production, I (Wort Production)." Acta Alimentaria 34(4): 373-380. Consequences of low assimilable nitrogen wort Low available nitrogen, especially in very high gravity wort, below a threshold of around 150mg/L, may lead to “stuck” fermentations Amino acid uptake by brewing yeast • GROUP A (fast) Glu, Asp, Asn, Gln, Ser, Thre, Lys, Arg • GROUP B (intermediate) Val, Met, Leu, Isoleu, His • GROUP C (slow) Gly, Phe, Tyr, Try, Ala, NH3 • GROUP D (little or no) Pro NOTE: A variety of transport permeases exist for (active) amino acid uptake by brewing yeast Yeast growth vs. Diacetyl Levels 30 Valine/Isoleucine synthesis required required Synthesis of valine/leucine α-acetolactate produced acetolactate produced Degrees Plato Yeast growth g/L Diacetyl (mg/L) 0.9 0.8 0.7 20 0.6 0.5 15 0.4 10 0.3 0.2 5 0.1 0 0 0 1 2 3 4 5 Days Fermentation 6 7 Diacetyl Yeast Growth 25 1 Mechanisms for yeast food enhanced reduction of diacetyl levels? Oxidative Decarboxylation α- acetolactate By-product of valine and isoleucine synthesis by yeast Yeast reductase - fast diacetyl acetoin (Slow) Requires functioning cell membrane to allow for uptake of diacetyl prior to reduction Provision of usable nitrogen source Amino acids supplied by yeast foods plus other nutrients results in a more May reduce requirement for synthesis of valine/isoleucine (and thus α- acetolactate vital cell that is enhancing diacetyl uptake and reduction? and diacetyl production)? Yeast nutrition potential problems for fermentation • Spectrum/availability of wort sugars – e.g. High glucose worts may slow fermentation • Insufficient FAN – Stuck fermentation, change in flavour profile • Metal ion availabitiy – e.g. Low Zn, excess Ca, insufficient Mg, toxic Cu • Vitamin deficiency (e.g. pantothenate - sulphur off-notes) • Stressed yeast - impaired nutrient uptake (ethanol, pH, CO2 , temp., starvation, osmotic pressure….) Yeast Foods Yeast “foods” Multifunctional roles • Alleviate CO2 inhibitory effects • Extra sources of assimilable nitrogen (hydrolysed protein) • Extra sources of vitamins • Extra sources of metal ions (to increase bioavailability) Commercial Yeast Foods Available Components • Amino acids (FAN)- as protein hydrolysate or added as individual amino acids • Low molecular weight peptides • NH4+ • Growth factors (vitamins) • Protein (non-utilisable) – active in CO2 nucleation? • Sulphur/Phosphorous • Zn/Cu/Mn/Ca (enzymatic and structural roles) • Yeast ‘hulls’ (broken cell walls) as sterol/FA source, nucleation site • Ergesterol – expensive Typical Yeast Food Effects* • Improved alcohol yield (increased extract utilization) • Reduction in fermentation time (12-24h) • Enhanced yeast vitality – Protect against stress • Enhanced diacetyl removal/consistent diacetyl specification • Control of undesirable flavour compounds * Depending on the yeast food composition Nutrient Trial Results Eastern European Brewery A 98 100 Control 90 80 Nutrient 71 70 60 50 34.9 33.2 40 30 20 6.8 6.3 10 0 Primary Fermentation Time (days) VDK in specification at time (%) Average Diacetyl (ppb) Nutrient Trial Results Eastern European Brewery B Control 160 148 140 Nutrient 120 87 100 80 60 40 4 20 6 3.7 3.3 0 Primary Fermentation Rate Final Diacetyl Level (ppb) (degP/Day) Residual Extract Servomyces Weihenstephan Trials 15L lab trials • • • • • • • Sample 1: no zinc addition (O-wort) Sample 2: addition of 0.3 mg/L ZnCl2 Sample 3: addition of 8 mg/L inactive yeast Sample 4: addition of 8 mg/L SERVOMYCES Sample 5: addition of 2.3 mg/L SERVOMYCES Sample 6: addition of 8 mg/L inactive yeast + 0.3 mg/L ZnCl2 Initial yeast zinc pool: 4.1 mg/100 g 0-wort ZnCl2 inactive yeast SERVOMYCES High Addition SERVOMYCES Low Addition inactive yeast + ZnCl2 Initial wort [mg/L] 0.10 0.35 0.11 1.22 0.36 0.34 Crop yeast [mg/100 g] 2.67 4.15 2.63 11.61 5.13 3.11 Servomyces Weihenstephan Trials Fermentation in 15L CCT at 10 ºC 14 ZnCl inactive yeast Servomyces - high addition 0-Wort Servomyces - low addition inactive Yeast + Zn Cl Extract [°Plato] 12 10 8 6 4 2 0 1 2 3 4 5 fermentation days 6 7 8 9 Servomyces Trial Results US Brewery 14 13 12 Servo addition A Servo addition B No Servo A No Servo B Extract [B rix] 11 10 9 8 7 6 5 4 0 1 2 3 Ferm entation days 4 5 6 Propagation • With each cell division the internal zinc pool is divided between mother and daughter cell • Fermentation 4-5 times biomass increase • Propagation 15-20 times biomass increase => very low internal zinc pool Slower, less efficient propagation Fermentation problems in 1. generation beer Slower, stuck fermentation Longer diacetyl rest Less flocculent yeast The Impact of Nutrients on High Gravity/Adjunct Brew • 60% malt / 40% adjunct / 18°P / Gen 1 18.0 Control A F B D C 16.0 Extract [% w/w] 14.0 E 12.0 10.0 8.0 6.0 4.0 2.0 0 1 2 3 time [day] 4 5 Serial Repitching • Repeated stress for the yeast • Decline in yeast activity • Resistance to stress is strain dependent • Contamination • Genetic drift/petite mutation The Impact of Yeast Generation on the Use of Nutrients High Gravity/Adjunct Brew 18.0 16.0 A F B D C Control B E 18.0 16.0 E A D F C 14.0 Extract [% w/w] 12.0 10.0 8.0 12.0 10.0 8.0 6.0 6.0 Gen 1 4.0 Gen 6 4.0 2.0 2.0 0 1 2 3 4 5 0 1 2 3 time [day] 16.0 Control A F B D C E 14.0 12.0 10.0 8.0 6.0 Gen 8 4.0 2.0 0 1 4 5 time [day] 18.0 Extract [% w/w] Extract [% w/w] 14.0 Control 2 3 4 5 6 7 time [day] 8 9 10 11 12 13 14 6 7 8 9 Nucleation Sites Effect of dissolved CO2 • Inhibits growth and metabolism • Decreases multiplication rate • Reduces biomass yield • Increases lag phase • Inhibition of nutrient uptake • Loss of cell division and bud formation Nucleation Sites • Particles working as nucleation sites reduce the solubility of CO2 • Most nucleation in a fermenter occurred from particles that sedimented to its bottom • Particle size is a likely determinant of the particle’s ability to be an effective CO2 nucleator • The more porous a particle is, the most efficient it is as a nucleation site • Soy flour may be ruled out due to allergen issues Nucleation Sites – Degassing Activity released CO2 after 2.5 hours 4 CO2 [g/L] 3 2 1 0 Control Soy Flour A B C Why Nutrients? • Sufficient supply of nutrients to the yeast • Predictable, fast and consistent fermentations without the formation of undesired flavours • Higher alcohol yields • High gravity brewing and increased amounts of adjuncts can result in slow or stuck fermentation • Insufficient nutrient supply will also result in formation of undesired flavours