Fermentation Control & Impact on Flavour STEPHEN NELSEN

Fermentation Control & Impact on Flavour STEPHEN NELSEN

RERUM COGNOSCERE CAUSAS Why do FV conditions affect flavour? What compounds are relevant? How does the brewer drive them? Could I be doing something better?

WHAT S FERMENTATION ANYWAY? Many times we see the following chemical reaction: This tells us two molecules each of ethanol and carbon dioxide are created from one of sugar, Or 2.06kg of sugars give us 1kg of ethanol and a kg of CO2. If it was such a simple chemical reaction, we could accelerate it without side-effects. BUT THAT S NOT ALL YEAST DOES

Yeast cells emerge from dormancy, respire and adapt their metabolism to the wort. They bud and multiply in biomass, you only pitch the mother cells. The daughters are better fermenters. This reproduction consumes time, nutrients, and energy, preferably aerobically. Image Source: http://www.microbiologyonline.org.uk/themed/sgm/img/slideshows/3.1.4_fungi_2.png)

For ideal budding the yeast wants; Constant cool temperature Low hydrostatic (top) pressure Moderate osmotic pressures (gravity 10 P) Low carbonation Moderate ph 5 FAAN (Free amino nitrogen) EAAs (Essential amino acids) Carbohydrate Oxygen

FOR EXAMPLE : GLYCOLYSIS Yeast splits sugars into two pyruvate molecules to get the energy, in the form of ATP (takes 2 ATP, produces 4 ATP : nets +2 ATP) In aerobic respiration, yeast gains much more energy from complete oxidation to CO2 and water. In fermentation, that s all the ATP energy the yeast gains Note acetaldehyde is a major intermediate before alcohol. It acts as final electron acceptor to oxidise NADH back to NAD + http://www.intechopen.com/books/food-industry/yeast-world-s-finest-chef

ACETALDEHYDE Only one step away from alcohol Can be excreted by unhealthy or stressed yeast Normally present in finished beer at about 3-10ppm, which is below average threshold. Acetaldehyde should be >95% of all aldehydes in beer, influential in staling. High Acetaldehyde in finished beer results from; Poor yeast health, Zn ++ deficiency, high pitching temperature, short maturation time infection by Zymomonas, oxidation of EtOH by O2

DON T SUFFOCATE YOUR FERMENT Early pressure application during primary fermentation will drastically increase acetaldehyde. As will fast fermentation, temperature increase, increased pitching rate, low wort oxygen, or infection. Image courtesy Geene King www.greeneking.co.uk

STRATEGY TO CONTROL ACETALDEHYDE Acetaldehyde excreted early, to 20 40 ppm, and can decrease to less than 10ppm in well matured beer, so taste your ferments. A warm rest at end of fermentation, not rushing. Ensuring yeast cultures are viable and vital Providing Zinc, especially if brewing from RO water Use of O2 not air Avoiding O2 post fermentation, and of course, Diligent hygiene.

THE CYSTEINE AND METHIONINE PATHWAYS Wort sulphates are consumed, reduced through sulphite, reduced through sulphide, incorporated into cysteine and eventually methionine. With insufficient energy, ATP, the cell will excrete SO 2, struck match aroma. With imbalanced nutrition, low FAN, the cell will excrete H 2 S, rotten egg gas

HIGHER ALCOHOLS (FUSELS) Yeast converts wort FAN by deamination, decarboxylation and reduction, into higher alcohols, also called fusels. Yeast forms other alcohols through hydroxyl and keto acids. Some higher alcohols are synthesised from sugars via acetate. Higher temp, stirring or pumping green beer, low FAN, repeated drauflassen, high gravity, warm pitching, all cause excessive fusels. Some fusels persist into the beer, leaving a burning solventy note. Higher alcohols esterify during conditioning

ESTERS Highly significant, determinative of aroma. Yeast strain has a fingerprint of ratios. Derived from the fatty acids or higher alcohols. Levels around 60 80 ppm OK, more than 2x fusels. Temperature is the biggest broadest lever the brewer has. Plentiful lipid and fatty acid during growth suppresses ester formation Less wort aeration -> slower fatty acid metabolism -> more esters. High O.G. > 13 P increases esters, but not proportionally to sales gravity.

VDKs: Vicinal di-ketones 2,3 butanedione and 2,3 pentane dione Neither is actually produced directly by yeast. In valine synthesis, yeast excretes acetolactate, which directly oxidises into diacetyl outside the cell in the beer. Yeast has high affinity for VDKs; absorbing them readily, and turns them into diols. Valine, and leucine, are part of FAN, total of which should be >180ppm.

EXCESS VDK LEVELS CAN BE DUE TO: High start of fermentation temperature -> rapid yeast growth -> too much acetolactate Variations in VDK levels occur due to yeast strain variances, Poor vitality, petites (respiratory deficient mutants) with poor membrane integrity, sluggish to reabsorb, Very high ph >5 prevents conversion, O2 on transfer converts more acetolactate Infection, particularly by Pediococcus, can flood the beer with diacetyl, and even ropy polysaccharides, a condition called sarcina

STRATEGIES TO REDUCE VDK SHOULD INCLUDE: Always plot the fermentation curve Warm rest end of primary fermentation: (Narziss rest) 24 hours contact 15 C Lower pitching temperature High FAN, Zn ++, Mg ++ Krausen green beer with working wort Maturation temperature regimes Monitoring the precursor, Acetolactate. If still present at packaging, oops, heat and oxygen in distribution convert more to diac. Cheapest test is to heat a sample to 60 for 2 hours, then cool & sniff vs control.

HEAT AND OTHER STRESSES Cells release HSP (heat shock proteins) Temperature abuse (rapid heating or cooling) Ethanol, as well as many other stress conditions. Sub-lethal heat, above 35 C, and ethanol stress, induced HSPs which had radical effects on protein efflux and the plasma membrane structure. [Piper 1995] Some HSPs have been shown to assist folding of other proteins and maintenance of membrane permeability, but flavour impacts are yet to be measured. This work, on molecular chaperones, is being continued by Kevin Morano and team at the Uni of Texas in Houston. Viz Verghese at al 2012. Image of HSP40, known as chaperone Dnaj from Wikipedia: https://en.wikipedia.org/wiki/chaperone_dnaj

DON T LEAVE YOUR BEER IN CONTACT WITH EVEN DORMANT YEAST TOO LONG, OR.. Proteolysis first disrupts and disperses metabolic processes Then cell wall rupture releases; nitrogenous compounds polysaccharides nucleic acids fatty acids C8 C16 vitamins heavy esters terpenes higher alcohols ethoxy-furans, etc.

THE VARIABLES AT YOUR FINGERTIPS

SOME SIMPLE ONES ALREADY MENTIONED Get Zinc to about 0.2 ppm FAN 140 180 ppm Gravity of wort Time in FV Vessel shape Shifting beer

EFFECT OF PITCHING RATE Low pitching rates result in long lag phase, increase higher alcohols, esters and diacetyl, and lower attenuation, as some sugars are not fully metabolized. Poor yeast growth can lead to high sulphur dioxide levels. On the other hand, sulphides are normally present, and too little will mean your beer is not true to type. High pitching rates develop off flavours, cells only multiply up to a certain peak concentration, so higher pitching rates lead to fewer doublings, less daughters. Pitching yeast should be slightly colder than the wort, not hotter. [Kunze 1999] Pitch inline to disperse yeast uniformly, avoid clumps, reduce wort oxidation, and get earliest contact between our beloved yeast and our precious wort. 1 million cells per ml per degree Plato: Ales slightly lower, lager somewhat higher.

OXYGEN Air saturated into 10 plato wort is only about 6 ppm O 2. 8 ppm is desirable. Beware of oxidation of wort and sensitive hop volatiles if oxygenating at the Heat Exchanger, and waiting till the FV is full before pitching, worse with dry yeast. Ideally pitch in line to allow the yeast to get the O2, not the wort. Oxygen solubility is inversely proportional to temperature and gravity, so higher temps and higher gravities, less solubility. Too much O 2 gives too vigorous fermentation and excessive yeast growth with consequent changes in beer flavour, at the expense of alcohol production [Lewis and Young 2002] Too little O 2 yeast vitality suffers, with attendant maladies.

EFFECT OF TEMPERATURE Some Lagers are pitched around 6 and rise to 8, after primary ferment reduced by 1 degree per day to 4 and moved to lager cellar. Ales pitch at 15 to 18 rise to 20 or 22 Belgian Saisons like Dupont are used up to 35 C (Wyeastlab) Ester production is proportional to temperature. Fusels, aldehydes, VDK, sulphurs all increase, but by less. Attemperate slowly, the consequences of temperature shock are shocking

EFFECT OF PRESSURE Shallow fermenters like the Yorkshire Square, the Burton Union, and most open top FVs produce quite fruity beers. Deep fermenters impose greater hydrostatic pressure Le Chateliers principle tells us this would decrease production of gases and volatiles, particularly esters. Higher pressures can reverse some of the effects of higher temperatures, and theoretically achieve lower esters and fusels at a given temperature.

KRAUSENING, DRAUFLASSEN, SKIMMING Addition of freshly fermenting wort to a beer near end of primary fermentation, introduces young active yeast. Krausening assists maturation because yeast has its maximum reducing power and metabolic rates during cell multiplication. Topping up (darauflassen) within 24 hours, infusion of fresh wort (not yeast) into a ferment. Avoid temperature shock, no lag phase. A loose, dirty brown layer on ferment after krausen has collapsed is called Schlaucherdecke and should be removed before transfer so that it does not sink down and contaminate the yeast. [Kunze 1999]

DIACETYL REST Yeast could remove ten times more diacetyl than it produced if healthy. ph affects extracellular conversion of acetolactate to VDKs, Ok above 4.2, but sour beers are likely to suffer preserved acetolactate etc. Precursors will take up oxygen and convert to VDKs if beer is aerated. Resuspending yeast by pumping, rousing or transfer favours diacetyl removal, which is another benefit to moving beer off lees. This rest day has been found to also improve aldehydic flavour and limit sulphidic flavours, so perhaps diacetyl rest is a mis-nomer?

CHILLBACK Avoid early chillback, or crash chilling. Successful lagering requires that the beer not be subjected to temperature fluctuations. It is important that the temperature is brought down gradually 1 to 3 C /day ) until the lagering temperature has been reached. Dropping the temperature too fast will inactivate the yeast, which could reduce the metabolizing of sugars and cause subsequent difficulties with diacetyl off-flavour [Goldamer 1999] Industrial breweries perform chillback through a heat exchanger Unitank method is prone to stratification

HARVEST AND SEPARATE YEAST FROM BEER Ale dropping or shifting a ferment after 24 48 hours removes trüb and dead yeast, and rouses the ferment. After chillback, ensure beer is not sitting on a plug of live yeast. Yeast in cone can warm up and exhibit hot spots, up to 10 C higher than the bulk. Brewers found very high pitching rates risked hot spots in the cone of FV leading to autolysis. [Knudsen, 1985]

I LIKE TO MOVE IT MOVE IT

DOWNSTREAM Get yeast out before dry hopping Chilling beer to 0 C or -1 C improves clarity and stability. Squat tanks enable quicker clarification Check VDKs before filtering.

WHEN THINGS GO WRONG Don t rush through fermentation or maturation on a account of demand. Only rouse with oxygen or sterile air in the first 24 hours. Be careful about allowing higher peak temperature to compensate for sluggish fermentation. Shifting beer from tank to tank may be better than rousing with CO 2 Take quick action, which may include krausening, dumping and brewing again with better yeast and better conditions. Just tipping in more dry yeast is usually ineffective.

FERMENTATION OFF FLAVOURS FLAVOUR Descriptor Possible causes Astringency Diacetyl Mouthpuckering Butterscotch Autolyzed culture yeast, thermal shock, alcohol. Incomplete fermentation, low FAN, early chillback DMS Overcooked corn Yeast converted DMSO Yeasty Vegemite Autolysis Fusel Alcohols Solventy, Vinous, lacquer Warm ferment, rousing

FERMENTATION OFF FLAVOURS /continued FLAVOUR Descriptor Possible causes Sulphitic, SO2 Struck match, sharp Poor growth, stress Hydrogen Sulphide Rotten egg stress/ poor growth, low FAN Other Sulfurs Rotten, putrid meat Poor yeast nutrition, shock Nonenal, long aldehydes Papery, stale Bitter Bad Taste in mouth Oxidized fatty acids and sulphitic reactions Sacked for not monitoring fermentation

SUMMARY AT PITCHING Ensure yeast #s, vitality, nutrition. Slightly cooler and well oxygenated 8ppm DURING FERMENT Daily attendance and plotting ph, gravity, taste. Drop tank bottoms, free rise in temperature. Don t spund too early END OF PRIMARY FERMENTATION FERMENTATION CONTROL FOR DUMMIES.. Monitor VDKs Separate beer and yeast Take your time

THANKS EVERYONE REFERENCES Fix, G 1989 Principles of Brewing Science, Brewers Publications, boulder Co Goldamer, Ted 1999 The Brewers Handbook KVP Publishers Knudsen, Finn B. 1985 Fermentation Symposium Part II Fermentation Variables and their control MBAA Technical Quarterly 22 (4) Kunze, Wolfgang Technology Brewing and Malting VLB 7 th Edition 1999 Lewis Michael J, and Young, Tom W, 2002 Brewing 2 nd Edn. Kluwer Academic, Plenum publishers Munroe, James H. 1995 Fermentation in Handbook of Brewing Edited by William A Hardwick, Marcel Dekker publisher Real 2 Real I Like to Move It CD Single 1994 Olaniran, Adeamola O., et al, Effects of fermentation temperatures on the composition of beer volatile compounds, organoleptic quality and spent yeast density. In Electronic Journal of Biotechnology www.ejbiotechnology.info. Vol 14 (2) March 2011 Piper, P.W. The heat shock and ethanol responses of yeast exhibit extensive similarity and functi9nal overlap in FEMS Microbiological letters, December 1995