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

• 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.

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

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

Species of Sulphur Dioxide

• Three forms exist1) Molecular SO2 SO2H2O2) Bisulphite HSO3

-

3) Sulphite ion SO32-

• Dependent on pH

• 1st and 2nd ionizations

HSO3-

The most predominant form we would see in brewing

18.7

86.1233

322

pKaSOHHSO

pKaHSOHOHSO

161412108642

180

160

140

120

100

80

60

40

20

0

Total Sulfur Dioxide ( mg/ L)

Lag

Tim

e f

or

EPR

(m

in)

Fitted Line PlotLag Time for EPR = 48.09 + 5.359 Sulfur Dioxide (SO2)

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

C O + HSO3- C

OH

SO-3

carbonyl Sulphur dioxide at beer pH Flavor neutral adducts

Free vs Bound Fractions

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

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)

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

Released in packaged goods

O-S

OOH

O

OH

O

O S O-

O

O

H2O

Formed during fermentation

O-

S

O

HO

OH O-

S

O

HO

O

trans-2 nonenal

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 SO2 additions 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.

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”

Sulphate Assimilation PathwaySO4

2-

3H+

3H+

SO42-

Cell Wall

LAST

HAST

APS

ATP

PPiSulphate Transporters

PAPS

ADP

ATP Sulphurylase

APS Kinase

HSO3-

NADPH, Trx (red)

PAPS ReductasePAP + NADP+ , Trx (ox)

Sulphite Reductase

6H+ + 6e-

6 H2O

S2-

Sulphite Pump

ATP

Toward cys –met-SAM biosynthesis

• 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

SUL

Family

Regulation of Sulphur Intermediates

• SAM Regulates numerous genes involved in Sulphate Assimilation Pathway

• Upregulation of SO42-

Transporters (SUL1)

• PAPS is extremely toxic so cellular regulation mechanisms exist to recycle PAPS (Met22p).

ASPARTATE

HOMOSERINE

THREONINE

O- ACETYL HOMOSERINE

HOMOCYSTEINE

ISOLEUCINE

METHIONINE

S-ADENOSYL METHIONINE

(SAM)

H2S

HSO3-

PAPS

APS

SO42- (Int)

SO42- (Ext)

Acetyl CoA

Threonine deaminase

Acetolactacte Pyruvate Lyase

ASP-K

OAH Sulphydrylase

ATP-S

ATP-K

HAST

SiR

Controlling SO2 - What is Known Today

Or + = If factor increases, SO2 increases Or - = If factor increases, SO2 decreases

Physical Mechanisms Likely InvolvedHSO3

- is produced cannot be reduced to H2S due to blockage in the metabolic flux

SO2 Excretion into Fermenting

Worts

PAPS

3-

NADPH, Trx (red)

PAPS ReductasePAP + NADP+ , Trx (ox)

Sulphite Reductase

6H+ + 6e-

6 H2O

S2-

PAPS

HSO3-

NADPH, Trx (red)

PAPS ReductasePAP + NADP+ , Trx (ox)

Sulphite Reductase

6H+ + 6e-

6 H2O

S2-

Sulphite Pump

Downstream Blockage

Wort DO ( )

Wort UFAs ( )

Ferm Temp (Ç)

Vitamins ( )

DP1/DP2 Ratio (+)

Wort OG(+)

Darauflassen ( )

Pitch Rate ( )

Viability ( )

Trub Solids( )

Glycogen Levels ( )

CO2 Back PSI(+)

Generation (+)

Yeast Strain

Wort Met & Cys

Mitochondrial Development ( )

Res Def Mutants

Mit ATP

Defects in ISC Formation

Sulphite Reductase Activity ( )

Wort pH (+)

HCO3- Inhibition of

Sulphite Reductase Activity ( )

MET5 b – Subunit SiR

MET10 (-) a – Subunit SiR

MET1 (-) – UPP-III transmethylase

SirohmeeMET8 (-) –

dehydrogenase / ferrochelatase

MET2(-) HOA-Transferase

SSU1 (+) – Sulfite Pump

Zinc

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?

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? Ergosterol

The Importance of Membrane Formation

• “If you screw up the membranes, you will screw up the performance of the yeast” Dr. Ryder, MillerCoors

O

O

P OO-

O

O

Non polar tail- Fatty Acids

Polar Head

Head Tail

Membrane

Sterols

Glycerol ester bond

Oleic Acid (C18:1)

O

PalmiticAcid (C16:0)

O

Membrane Bound Proteins

Lipid bilayer

X

Kink in tail due to double bond

Many enzymes/proteins that serve cellular metabolic roles are membrane bound and interact with the lipid constituents in the membrane

Trials Altering Oxygen

• 22 Full Factorial Design

• Wort DO drove downSO2

• So did WP Stand Time

1810

24

22

20

18

16

14

12

103010

DO

Mean

WP Stand

Main Effects Plot for Sulfur DioxideData Means

Trial Whirlpool Stand time

Wort Dissolved Oxygen

1 0 0 2 - - 3 + - 4 - + 5 + +

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

2001000

9

8

7

6

5

4

3

2

1

0

(Hrs)

(mg/

l)

Comparison of Total SO2 Development During Fermentation

Wort Streams 1-3-7-8

Wort Streams 2-4

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

Trials Fatty acid (mg/l) Macadamia nut

oil base wort Lipase treated mash trial

Palmitic acid, C16:0 1.04 2.71 Palmitoleic acid, C16:1 0.00 0.07 Stearic acid, C18:0 0.00 1.16 Oleic acid, C18:1 0.16 0.825 Linoleic acid, C18:2 1.04 3.31 Linolenic acid, C18:3 0.15 0.35

16.5

12.7

9.4

0

2

4

6

8

10

12

14

16

18

Control Macadamia nut oil Lipase Trial

SO

2 (

mg

/l)

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

20mg/L0mg/L

30.0

27.5

25.0

22.5

20.0

17.5

15.0

C18:2

Sulp

hur

Dio

xide

Individual Value Plot of Sulphur Dioxide vs Linoleic Acid Additions

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 !

Nucleation SDOTemperaturePitch Rate

29

27

25

23

21

SO

2-E

OF

Main Effects Plot (data means) for Total Sulphur Dioxide

Centerpoint

Internal Trials with Soya Flour in Tall Verticals

• DOE with 4 factors• One was Nucleation

Site aids • NSD

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

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)

(ppm)

-1.8-1.4-1.0-0.6-0.20.20.61.01.41.82.22.6

PC

2-LPC

1-LPCAPE

PA PEPG

DPG

(ppm)

-1.8-1.4-1.0-0.6-0.20.20.61.01.41.82.22.6

PC

2-LPC

1-LPCAPE

PA PEPG

DPG

2.3

19.7

3.41.2 1.9

68.7

13.6

3.4 3.8 3.42.03.0

5.06.0

1.0

17.0

4.02.0

5.0

10.0

0.0

10.0

20.0

30.0

40.0

50.0

60.0

70.0

80.0

Bra

ssic

aste

rol

Cam

pest

erol

Stig

mas

tero

l

D7

Cam

pest

erol

D7

Stig

mas

tero

l

beta

-sito

ster

ol

beta

-sito

stan

ol

Del

ta-7

-Ave

nast

erol

Del

ta-7

,25-

Stig

mad

ieno

l

Del

ta-5

-Ave

nast

erol

mg

/100

g

Other Lipids in Trub Solids – Phytosterols Predominant Barley Phytosterols are structurally

different than yeast sterols (ergosterol / lanosterol)

Free (shaded) Total (empty)Structurally very similar to ergosterol

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

H2N

HOOC

CH CH2CH2 S CH3

Methionine

H2N

HOOC

CH CH2SH

Cysteine

H2N

HOOC

CH CH2CH2 S

+CH2

S-Adenosyl Methionine (SAM)

CH3

O

NH2

N

N N

N

H

OH OH

H H

Met and SO42- Uptake Patterns in Clear vs Cloudy Worts

Cloudy GT Treated

0 50 100 150

0

5

10

15

Fermentation Time (Hrs)

SO

2 (

mg/l)

Cloudy GT Treated

0 50 100 150

0

10

20

30

40

50

Fermentation Time (Hrs)

Met

hion

ine

(mg/

l)

Cloudy GT Treated

0 50 100 150

215

225

235

245

Fermentation Time (Hrs)

Sul

phat

e (m

g/l)

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 ?????

Interpretation

• Downstream Blockage- Sulphite Reductase

• Putative Sulphite pump removes toxic HSO3

- from the cell

• SAM depletion leads to up regulation of SUL1 (SO4

2- uptake) gene

• More Sulphate is taken up by the yeast whilst sulphite is excreted out

SO42-

3H+

3H+

SO42-

Cell Wall

LAST

HAST

APS

ATP

PPiSulphate Transporters

PAPS

ADP

ATP Sulphurylase

APS Kinase

HSO3-

NADPH, Trx (red)

PAPS ReductasePAP + NADP+ , Trx (ox)

6H+ + 6e-

6 H2OS2-

Sulphite Pump

ATP

Toward cys and met biosynthesis and SAM

Sulphite ReductaseX

HSO3-HSO3-HSO3-

SO42-

SUL1

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

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.

Effects of additional glucose to wort on sulphite, acetaldehyde, glycerol, and osmotic pressure taken from Gyllang et al. (EBC 1989)

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

Other Factors Related to Yeast Growth

• Glucose effects on mitochondria development

• The role of the mitochondria on SO2 production 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

1. Inner 2. Outer 3. Cristae 4. Matrix

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

3025201510

25

20

15

10

5

0

Temperature (C)

SO

2 (

mg

/L)

Effect of Fermentation Temperature on SO2 Production

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

Propagation Effects

• Example from a MillerCoors brewery

• Conditioning Effect?

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 ……

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.

Restricted Yeast Growth

1. 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

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

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

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

Theoretical Aspects - Yeast Sulphite Reductase (SiR) Structure

• Kobayashi & Yoshimoto (1982)

• Saccharomyces cerevisiae Sulphite Reductase is a 604 kDa protein with an a2b2 structure and also contains different prosthetic groups

• The 116 kDa a subunit is encoded by the MET10 Gene

• The 167 kDa b subunit is encoded by the MET5 Gene

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

S

FeS

Fe

Fe

SFe

S

N

N

N

N

H

H

HO

O HO

O

OH

O

OH

O

Fe2+

OH

O

HOO

HO

O

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 a8b4 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

How HSO3- is Reduced

S

OH O

-O

S

S

S

S

Fe Fe Fe

Fe

N N

NN

Fe2+

S

1+

IIHSO3-

Activated water molecules (Solid circles)

III) Bi-Sulphite binds to Fes through its sulphur

H+

H2O

S

O-

-O

S

S

S

S

Fe Fe Fe

Fe

N N

NN

Fe3+

S

2+

III

IIIII) Delivery of a H+ to protonated oxygen with concurrent transfer of 2e- through ISC & Siroheme releases water and results in dioxygenated sulfur

S

S

S

S

Fe Fe Fe

Fe

N N

NN

Fe2+

S

1+

I

I) Two e- reduce cofactors and prime catalytic site for HSO3

- binding

Key Point: No intermediates are released during HSO3

- reduction

to S2-

How HSO3- is Reduced

IIIIV) & IVV) Two electrons are delivered through cofactors and two protons are delivered to the bound substrate intermediates with release of H20 at each step

S

O-

-O

S

S

S

S

Fe Fe Fe

Fe

N N

NN

Fe3+

S

2+

III

2e- + 2H+ 2e- + 2H+

H2O

H2OS

O

S

S

S

S

Fe Fe Fe

Fe

N N

NN

Fe3+

S

2+

2-

IV

S

S

S

S

Fe Fe Fe

Fe

N N

NN

Fe3+

S

S2-

V

2+

V) Sulfide is poor siroheme ligand and dissociates from the catalytic site upon a proton transfer

HS-

Toward homocysteine formation

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

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

Questions ?

Email: eric.samp@millercoors.com