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PRACTICAL AND THEORETICAL ASPECTS OF SULPHUR DIOXIDE PRODUCTION AND CONTROL

MBAA Out of Town Meeting, Grand Lake, CO Sept 25, 2010. PRACTICAL AND THEORETICAL ASPECTS OF SULPHUR DIOXIDE PRODUCTION AND CONTROL. Eric Johann Samp, Ph.D. MillerCoors. Abstract.

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PRACTICAL AND THEORETICAL ASPECTS OF SULPHUR DIOXIDE PRODUCTION AND CONTROL

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  1. MBAA Out of Town Meeting, Grand Lake, CO Sept 25, 2010 PRACTICAL AND THEORETICAL ASPECTS OF SULPHUR DIOXIDE PRODUCTION AND CONTROL Eric Johann Samp, Ph.D. MillerCoors

  2. Abstract • Sulphur dioxide is an important metabolite formed by lager yeast during fermentation and is a critical parameter in beer to control for various reasons (flavor, flavor stability, physical stability, and legal requirements to name a few). This presentation will review the biochemistry of SO2 formation by yeast and discuss what is known today on how to control sulphite production. These include wort oxygenation, wort clarity and arguments on nutritional vs physical effects of solids in fermentation, lipids and which ones and perhaps why, fermentable carbohydrates, wort FANs and S-amino acids, zinc, fermentation temperature, yeast pitch rates, and yeast health. For the craft brewer, simple diagnostic tools will be discussed as possible ways to trouble shoot what could be driving SO2 production. Finally, some theoretical views on why sulphur dioxide is produced by yeast will be presented.

  3. Agenda • What are the forms of SO2 • Why is SO2 control important for the brewer (good, bad, & ugly)? • How is SO2 formed by yeast • What is known today on how to control SO2 • Trouble shooting guidelines • Some theoretical views on why SO2 is produced by lager yeast during fermentation

  4. Question • Raise your hand if you can measure SO2 today in your operations? • How many can measure H2S? • Samples Spike with Sodium Bi-Sulphite flavor standards - NOSE ONLY: Please do not ingest - You should pick up either a burnt match or vitamin pill bottle like aroma

  5. HSO3- The most predominant form we would see in brewing Species of Sulphur Dioxide • Three forms exist1) Molecular SO2 SO2H2O2) Bisulphite HSO3-3) Sulphite ion SO32- • Dependent on pH • 1st and 2ndionizations

  6. OH HSO3- C + C O SO-3 Sulphur dioxide at beer pH carbonyl Flavor neutral adducts Why Worry About SO2 Control – The Good • Powerful Antioxidant: - Protects isohumulones from free radical attack - Recent report on quenching free radicals from malts that survive fermentation(Cortes et al., ASBC) - Close relationship with EPR Lag Time • Flavor Stability- Binds to flavor active carbonyls to render them flavor neutral

  7. Free vs Bound Fractions • Most of SO2 formed during fermentation is bound to aldehydes / carbonylsstudy courtesy of David Ryder – Miller Brewing Co.

  8. Why Worry About SO2 Control – The Good • Improves Colloidal Stability:- both bound (acetaldehyde) & free forms (Kaneda et al., 1994) • Antimicrobial agent (ATP Depletion) - Molecular SO2 is the most powerful but bi-sulphite can also be effective in free form - Molecular SO2 is the only form that is allowed to traverse across membranes (passive transport) - ATP Depletion through adduct formation with glyceraldehyde-3-phosphate (Hinze & Holzer, 1986)- ATP Depletion through inactivation of glyceraldehyde-3- phosphate dehydrogenase (Schimz & Holzer, 1979)- Sulphitolysis of Thiamine, reactions with methionine and pyridoxal phosphate (Rose, 1987)

  9. Formed during fermentation trans-2 nonenal Released in packaged goods Why Worry About SO2 Control – The Bad • Reversible binding with Aldehydes during fermentation rendering them inaccessible to yeast during fermentation- eg trans-2-nonenal (papery) can bind to HSO3- • Adducts dissociate in packaged goods upon oxidation causing premature staling

  10. Why Worry About SO2 Control – The Bad • Fresh Beer Flavor - Descriptors (Burnt match, Vitamin Pill Bottle) • If yeast are excreting SO2, they are “unhappy” and other off flavors follow • Reports on SO2additions to cask ale leading to H2S issues • Reports on detrimental effects on foam stability With permission from the Editor (Tom S.) of the Tech Q.

  11. Why Worry About SO2 Control – The Ugly • Allergenic Properties & other health related issues (Taylor et al., 1986)a) Induction of asthma in asthmatics (early 1980s) b) Throat & Gastrointestinal irritation, c) anaphylatic shock, d) hypochromic anemia (lack of energy, low iron, green skin), e) urticaria (skin rash) & pruritis (itching)) • Humans detoxify the body of free sulphite through sulphite oxidases found in the liver and kidney, which produces sulphate and is excreted out of the urinary tract.- severe deficiency in Sulphite Oxidases in individuals have lead to problems with dislocated occular lenses and severe neurological abnormailities • Legal Requirements in the U.S.- 10 mg/L max, if exceeded requires label declaration “contains sulphites”

  12. Cell Wall SO42- 3H+ 3H+ LAST HAST ATP ATP Sulphurylase SO42- Sulphate Transporters PPi APS ATP APS Kinase ADP NADPH, Trx (red) PAPS Sulphite Pump PAPS Reductase PAP + NADP+ , Trx (ox) HSO3- 6H+ + 6e- SulphiteReductase 6 H2O S2- Toward cys –met-SAM biosynthesis Sulphate Assimilation Pathway SUL Family • SO42- is the sole source of sulphite from yeast • It is utilised for the synthesis of s-aminoacids and other inorganic sulphur compounds • HSO3- is a toxic intermediate.Yeast have basal tolerance. Protective mechanismsexist to remove HSO3- • Any disruption in the pathwayafter sulphite formation couldconceivably lead to efflux

  13. Regulation of Sulphur Intermediates • SAM Regulatesnumerous genes involved in Sulphate Assimilation Pathway • Upregulation of SO42- Transporters (SUL1) • PAPS is extremely toxic so cellular regulation mechanisms exist to recycle PAPS (Met22p). SO42- (Ext) HAST SO42- (Int) ATP-S APS ATP-K ASPARTATE ASP-K PAPS HSO3- Acetyl CoA SiR HOMOSERINE H2S O- ACETYL HOMOSERINE THREONINE OAH Sulphydrylase Threonine deaminase HOMOCYSTEINE Acetolactacte Pyruvate Lyase METHIONINE S-ADENOSYL METHIONINE(SAM) ISOLEUCINE

  14. Controlling SO2 - What is Known Today  Or + = If factor increases, SO2 increases Or - = If factor increases, SO2 decreases

  15. Principal #1: Biomass Formation • Well established that a negative correlation exists between the amount of yeast produced during fermentation and SO2 production • Therefore any factors that promote yeast growth will support lower SO2 production • Wort Dissolved Oxygen - What do yeast use O2 for?

  16. Ergosterol The Role of Oxygen • Synthesis of Heme which serves as a prosthetic group for various cytochromes involved in lipid biosynthesis • Formation of UFAs - one mole of O2 for one mole of C16:1 or C18:1 • Synthesis of Sterols- 9 moles of O2 for one mole of ergosterol • Why are these important?

  17. X Oleic Acid (C18:1) Non polar tail- Fatty Acids Glycerol ester bond Kink in tail due to double bond Polar Head O O O PalmiticAcid (C16:0) O O- P O O Tail Head O Sterols Membrane Bound Proteins Membrane Lipid bilayer The Importance of Membrane Formation • “If you screw up the membranes, you will screw up the performance of the yeast” Dr. Ryder, MillerCoors Many enzymes/proteins that serve cellular metabolic roles are membrane bound and interact with the lipid constituents in the membrane

  18. Trials Altering Oxygen • 22 Full Factorial Design • Wort DO drove downSO2 • So did WP Stand Time

  19. Role of Wort Clarity: Lipids & Trub • Cloudy worts have been reported to off set SO2 production • Trub solids carried over from hot break separation leads to cloudy worts • Various lipids originate from brewing materials (malted barley, cereal adjuncts, hops) • Yeast will take up lipids (UFAs, PLs, LPLs Sterols) from wort and store/utilize them for membrane synthesis

  20. Development of SO2 for Varying Levels of Cloudy Worts • Wort streams 2-4 had very cloudy due to differences in wort production processes • High glucose wort fermentation • Both wort DO levels 10 mg/L • Trials Comparing Macadamia nut oil supplements and Lipase Treated mash

  21. Trials

  22. Unsaturated Fatty Acids • Ohno & Takahashi (1987- Kirin) - C16:1 & C18:1 supplements lowered SO2- C16:0 had no impact • Samp & Hughes (EBC-2009) - Significant effect of C18:2 - No effect of yeast 16 or 18 Carbon UFAs or SFAs • Samp & Hughes (EBC-2009) - Also found some other phyto-lipids that could be affecting SO2 production

  23. Trub Solids – Physical or Nutritional • The addition of solids to fermentations has shown a stimulatory role in fermentation - (Bishop & Whitley, 1938 – “Flocculum”) • Groat & Ough (1978) in wine fermentations- DE, Talc & Grape solids stimulatory • Numerous authors have reported NO EFFECT ON SO2 from yeast foods that provide nucleation sites (soya flour) yet people still claim this !

  24. Internal Trials with Soya Flour in Tall Verticals • DOE with 4 factors • One was Nucleation Site aids • NSD

  25. Trub Solids – Physical or Nutritional • Originally proposed by Gyllang et al (EBC 1989) that HCO3- competes with HSO3- for active site in Sulphite Reductase • Samp & Hughes – EBC 2009 - Argument against HCO3- inhibition due to planar geometry of bi-carbonate • Karl Siebert (1986) – “DE Filtration of Wort” – CO2 inhibition of yeast growth- One solid studied was Lecithin (Phosphatidyl Choline) which is a barley phospholipid

  26. Other Lipids in Trub Solids- Phospholipids • Phospholipids from malted barley are similar – FA acyl constituents differ in the degree of unsaturation (more C18:2 / C18:3 vs C18:0 / C18:1) • Profiles trub solids using 31P NMR Spectroscopy • Phosphatidyl choline and lyso-phosphatidyl choline (LPCs)

  27. Structurally very similar to ergosterol Other Lipids in Trub Solids – Phytosterols Predominant Barley Phytosterols are structurally different than yeast sterols (ergosterol / lanosterol) Free (shaded) Total (empty)

  28. H2N NH2 CH2 CH3 CH2 S CH N N N N HOOC CH3 H2N CH2 CH2 CH2 S+ CH O S-Adenosyl Methionine (SAM) HOOC H H H OH OH Methionine H2N SH CH2 CH Cysteine HOOC Patterns in Met and SO42- Uptake • Cysteine is very low in worts • Methionine is available in measurable quantities and is transported via GAP and Met Permeases • Sulphate is only taken up throughpermeases when cell is deplete of Met, Cys, and SAM • SO42- assimilation pathway is energy (ATP) demanding

  29. Met and SO42- Uptake Patterns in Clear vs Cloudy Worts 1. SO2 production started within 48 hours for the clear worts 2. Met uptake initiated immediately but faster in cloudy worts – all was consumed 3. Sulphate uptake occurred after 50 hours of fermentation in cloudy worts but immediate in the clear worts ?????

  30. Sulphite Pump X SulphiteReductase Interpretation SO42- SO42- Cell Wall 3H+ • Downstream Blockage- Sulphite Reductase • Putative Sulphite pump removes toxic HSO3- from the cell • SAM depletion leads to up regulation of SUL1 (SO42- uptake) gene • More Sulphate is taken up by the yeast whilst sulphite is excreted out 3H+ SUL1 LAST HAST ATP ATP Sulphurylase SO42- Sulphate Transporters PPi APS ATP APS Kinase ADP NADPH, Trx (red) PAPS PAPS Reductase PAP + NADP+ , Trx (ox) HSO3- HSO3- HSO3- HSO3- 6H+ + 6e- 6 H2O S2- Toward cys and met biosynthesis and SAM

  31. Other Factors Related to Yeast Growth • Zinc:- if too low, we know we can see sluggish fermentations- Yeast alcohol dehydrogenase which catalyzes the reduction of acetaldehyde to alcohol is a zinc-metallo enzyme - If acetaldehyde builds up intracellularly, then it can bind directly with HSO3- - Once bound, the bi-sulphite cannot be reduced and is likely excreted out of the cell • Personal experience: Zinc is not the most significant control parameter

  32. Effects of additional glucose to wort on sulphite, acetaldehyde, glycerol, and osmotic pressure taken from Gyllang et al. (EBC 1989) Other Factors Related to Yeast Growth • Carbohydrate Profile (DP1/DP2 Ratio) - A common complaint is that using commercial adjuncts with high glucose (DP1) levels leads to SO2 problems. Argument was that increased osmotic pressure caused higher levels of glycerol, which inhibits EtOH dehydrogenase, which would lead to more acetaldehyde and pyruvate, which would bind to bisulphite

  33. 1. Inner 2. Outer 3. Cristae 4. Matrix Other Factors Related to Yeast Growth • Glucose effects on mitochondria development • The role of the mitochondria on SO2production was proposed by O’Connor-Cox et al (1993) at SAB - Studies blocking Mt ATP • Samp & Pratt (2010) have proposed that development of a mitochondrial lipid, cardiolipin, is involved

  34. Fermentation Parameters – Temp & Pitch Rate • Fermentation Temperature- Brewer & Fenton (1980) reported optimum around 16 ºC • Similar reports byNordlov(1985 EBC) • Pitch Rate- under-pitchingleads to increased SO2

  35. Principal #2 – If you treat your yeast poorly you will pay for it! • Low Viability & Vitality - Factors / practices that lead to low viability can lead to elevated SO2. 1) Holding yeast on green beer for extended periods (shut downs) then harvesting 2) Storing yeast too warm (<4 ºC)3) Storing yeast too long (> 48 hours)4) Improper mixing 5) Cropping practices (what to crop/waste)? • High Generations - Appears to be consensus - Personal belief is this not as critical as widely accepted • Repitching yeast with history of previous fermentations being high

  36. Propagation Effects • Example from a MillerCoors brewery • Conditioning Effect?

  37. What if you have SO2 issues? • Legal requirement (10 mg/L max) • If packaged you have 3 options:1) Re- label with a declaration ($$$)2) Store – Ilett & Simpson reported half life of 6 months at 25 ºC3) Do nothing (and pray!) • If in-process beer- CO2 purging WILL NOT strip out SO2- Dilution is the solution ……

  38. Trouble Shooting • What if you cannot measure SO2 but suspect it is high based on sensory? - assume H2S levels are acceptable • Relationship to biomass production- check records on total yeast cropor cell counts during fermentation- if lower than normal, then likely have a problem- Check EOF pH levels as this can correlate to yeast growth. If pH is higher than normal, then likely you have restricted yeast growth which could imply elevated SO2 • pH decline rate during fermentation can be a marker if you are experimenting in reducing SO2.

  39. Restricted Yeast Growth • Audit wort DO - check with portable orbisphere close to FV - restore to normal levels audit injection system: - plugged diffusors - leaking air lines - consider increasing if you need to react quickly - check if you are getting bubbles breaking out Video of O2 bubbles at manifold close to FV

  40. Restricted Yeast Growth 2) Check Yeast Growth Factors • Check wort clarity / trub solids carryover - Is the wort too clear? - Cloudy up worts – be careful (reduce vorlauf times, WP draw off rates, reduce wort settling times, cut back on kettle fining agents). - Lets blame the malt! Under-modified could imply less free UFAs Check CofA’s • Verify wort Zn levels - If wort pH has shifted up, then likely Zn levels have decreased

  41. Fermentation / Yeast Parameters • Check viability record (if available). Discard yeast with low viability or ones prone to sulphite production • Are you using a high generation yeast? - if > 10 then consider discarding • Audit yeast brinks for - proper temperature control - proper mixing • Are you storing yeast under green beer too long and too warm? - consider modifying cropping practices

  42. What if H2S and SO2 are both elevated? • Implies very unhappy stressed yeast!- All guidelines above apply • Suggests a blockage in the SO42- assimilation pathway past H2S formation so both intermediates are being excreted - realize flavor thresholds: SO2 is ppm & H2S is ppb • Similar problems exist in enological fermentations - Their solution is Nitrogen - Vitamin B - Zn based yeast foods

  43. Theoretical Aspects - Yeast Sulphite Reductase (SiR) Structure • Kobayashi & Yoshimoto (1982) • Saccharomyces cerevisiae Sulphite Reductase is a 604 kDa protein with an 22structure and also contains different prosthetic groups • The 116 kDa  subunit is encoded by the MET10 Gene • The 167 kDa  subunit is encoded by the MET5 Gene

  44. Prosthetic Groups of SiR • 2 Nucleotides (FAD and FMN) • A tetrahydroporphyrin called Siroheme • A [4Fe-4S] Iron Sulphur Cluster (ISC) bridged by a cysteine thiolate to Fes on proximalside of Siroheme • Siroheme & [4Fe-4S] cluster are a coupledelectronic unit involved in the 6 electron “Push-Pull” transfer to bound HSO3- for sulphite reduction

  45. Reduction of HSO3- to S2- • Characterization of Assimilatory SiR has been conducted in E. coli. • E. coli has the same prosthetic groups:Siroheme and [4Fe-4S] clusters but differs in its 84 structure. • It appears that the catalytic site in sulphite reduction occurs on the distal end of the siroheme and [4Fe-4S] ISC cluster. • Bisulphite’s sulfur binds to siroheme’s Fes • 6 Electrons must be transferred to S-atom of HSO3- for full reduction to Sulphide

  46. Activated water molecules (Solid circles) I III N N II HSO3- H+ H2O Fe2+ N N S 1+ -O OH O IIIII) Delivery of a H+ to protonated oxygen with concurrent transfer of 2e- through ISC & Siroheme releases water and results in dioxygenated sulfur III) Bi-Sulphite binds to Fes through its sulphur I) Two e- reduce cofactors and prime catalytic site for HSO3- binding S N N N N Fe3+ Fe2+ N N N N S S 1+ -O O- S Fe Fe Fe S S S S S S S S S Fe Fe Fe Fe Fe Fe Fe Fe Fe S S S 2+ How HSO3- is Reduced Key Point: No intermediates are released during HSO3- reduction to S2-

  47. 2- O III S H2O 2e- + 2H+ IV 2e- + 2H+ H2O 2+ HS- Toward homocysteine formation S2- V N N N N N N Fe3+ Fe3+ Fe3+ 2+ N N N N N N S S S -O O- S Fe Fe Fe S S S S S S S S S Fe Fe Fe Fe Fe Fe Fe Fe Fe S S S 2+ How HSO3- is Reduced V) Sulfide is poor siroheme ligand and dissociates from the catalytic site upon a proton transfer IIIIV) & IVV) Two electrons are delivered through cofactors and two protons are delivered to the bound substrate intermediates with release of H20 at each step

  48. So How is the Mitochondria Related? • Siroheme is partially synthesized inside the mitochondria • 4Fe-4S clusters are also synthesized in this organelle and exported out - Both synthesis and exporting require Mt ATP • Mitochondrial membrane lipids could be involved - Sterols - Cardiolipin

  49. Concluding Remarks • SO2 plays a critical role in the quality of beers - negatives & positives  process control. • Oxygen and Lipids are critical drivers on SO2 production • Yeast health is also a driver - treat your yeast properly

  50. Questions ? Email: eric.samp@millercoors.com

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