f15 industrial mycology

BREAD, WINE AND BEER – CENTURIES OF INDUSTRIAL MYCOLOGY

Microorganisms involved in winemaking

YeastSaccharomyces cerevisiaeSaccharomyces bayanus

Alcoholic fermentation• Converts sugars to ethanol• Flavour production

Malolactic fermentation• Converts malic acid to lactic acid• Flavour production

Lactic acid bacteriaOenococcus oeni

Harvest Crush Alcoholic Fermentation:

1. Spontaneous, or

2. Inoculated fermentation (Yeast addition), or

3. Combination of 1 & 2

Skin contact:

1. No – white wines

2. Yes – red wines

Ferment to completion

Malolactic Fermentation:

1. Spontaneous, or

2. Inoculated fermentation (Lactic acid bacteria)

Racking off leesPost-fermentation processing and bottling

Wine production: Microorganisms associated with grape berries

• Bacteria– Wine-related lactic acid bacteria– Acetic acid bacteria

• Yeast– Kloeckera/Hanseniaspora, Candida spp., Saccharomyces,

Pichia, Kluyveromyces, Brettanomyces/Dekkera, etc.– Saccharomyces present at extremely low counts on

surfaces of healthy berries– Much higher frequency on damaged berries

• Molds– Botrytis cinerea (the “noble rot”), Aspergillus (ochratoxins),

Penicillium, Mucor

Yeast & Vinification

• Saccharomyces cerevisiae (“sugar fungus from beer”); Saccharomyces bayanus– S. cerevisiae var. bayanus

• “Indigenous” yeast– Microflora of grapes– Initial fermentation

• Inoculated fermentations– Active Dry Yeast (Commercially produced yeast)– 2x106 cells/mL; Typically six cell replications

Major constituents of grape juiceCompound Percent

Water 76%GlucoseFructose

8-13%8-13%

Tartaric acidMalic acidCitric acid

0.2-1.0%0.1-0.8%

0.01-0.05%Tannins (catechol, chlorogenic acid, caffeic acid) 0.01-0.1%

Nitrogenous compounds (amino acids and proteins) 0.03-0.17%

Other minerals (phosphates, sulphates) TracesB-group vitamins (thiamine, riboflavin, pyridoxine,

nicotinic acid)Traces

Ascorbic acid TracesVolatile aroma constituents Traces

Colour constituents Traces

Fermentation stresses• Osmotic stress

– 22% (w/v) sugar; Aw = 0.982– 40% (w/v) sugar; Aw = 0.939

• Nutrient limitation– Nitrogen; Micronutrients

• Ethanol toxicityImplications• Stuck/sluggish fermentations

– Delay in sugar utilization; affects product quality• Off-flavour production• Spoilage

• Efficiency– Winery productivity– Financial losses

Log of Absorbance = yeast cell density

Increasing EtOH concentrationRapid oxygen depletion

Nutrient depletion

In Boulton et al. (1998)

Typical wine fermentation

Grape must • 20-26% (w/v) sugar (Table wine)• >35% (w/v) sugar (Icewine)

Acetic acid Acetate

Areas of wine yeast development• Stress tolerance

– Ethanol tolerance– Nutrient limitation

• Fermentation vigor– Effective nutrient utilization– Interactions/competition with other microbes

• Efficient fermentation at different temperatures• Consistent production of desired flavour and aroma

metabolites• Decreased production of undesirable by-products• Accelerated aging of wine (eg. Sparkling wine

production) - my MSc. topic!

Sparkling wine production

Yeast autolysis

• A lytic and irreversible process resulting from the release of intracellular yeast enzymes

• “post-death” • Occurs during wine aging• lengthy process (9-12+mo)• Results in release of cell

compounds which contribute positively to sparkling wine quality

Yeast autolysis in sparkling wine

Autolysis compounds

Origin Characteristics

Nitrogenous: proteins, peptides, amino acids

Yeast cell content Organoleptic, foam quality

Polysaccharides and mannoproteins

Yeast cell wall, grapes Organoleptic, foam quality, wine stability

Lipids Yeast cell content Organoleptic, foam quality

Nucleic acids Yeast cell content Organoleptic

Attempts to accelerate autolysis• Increasing temperature

• Increases enzyme activity• Undesirable off flavours – toasty,

excessively yeasty• Adding autolysed yeast

• Same undesirable flavours• Mix of killer/sensitive strains

• Some yeasts produce K2 killer toxin, which is toxic to many yeasts

• Impractical – most wine yeasts are “killers”

Martinez-Rodriguez, A., Gonzalez, R., & Carrascosa, A. (2004). Morphological Changes in Autolytic Wine Yeast during Aging in Two Model Systems. Journal of Food Science, 69(8).Todd, B., Fleet, G., & Henschke, P. (2000). Promotion of autolysis through the interaction of killer and sensitive yeasts: potential application in sparkling wine production. Am. J. Enol. Vitic., 51(1), 65–72.

Attempts to accelerate autolysis• Genetic approaches

• Most have centred on autophagy (“self-eating”)• Overexpression, knockdown or deletion of genes

Beer production

• Barley sugars: • Glucose, fructose,

maltose, sucrose, maltotriose, dextrins

• Beer wort (unfermented beer) is much higher in nitrogen and micronutrients than grape juice

• Higher inoculation rate (~107 cells/mL) than wine

Yeast in beer

FunctionANDFlavour

Yeast in beerImportant characteristics: • Fermentation vigour (how fast it ferments)• Attenuation (what percentages of the sugars it can ferment)• Temperature range • Flavours

• Esters – eg. isoamyl acetate (banana), ethyl caprylate (pineapple), ethyl caproate (apple)

• Phenols – eg. 4-vinylguiacol (clove), 4-ethylphenol (“barnyard”)

• These can be positive or off-flavours depending on beer style or amount!

• Off flavours – acetaldehyde (green apple), diacetyl (butterscotch), autolysis (in beer anyway!)

Why do yeasts produce flavours? • Classical model: these compounds are just a byproduct of metabolism• Recently: flavour production confers an evolutionary advantage!

Christaens et al. (2014) Cell. The Fungal Aroma Gene ATF1 Promotes Dispersal of Yeast Cells through Insect Vector

Atf1 = alcohol acetyl transferase

Beer production: Microorganisms

• Bacteria– Lactobacillus, Pediococcus, Acetobacter– Almost always NOT something that should be present in

beer/wort (unfermented beer) – Except for sour beer styles

• Lactobacillus and/or Pediococcus• Yeast

– Saccharomyces cerevisiae – “ale yeast”, “top-fermenting”– Saccharomyces pastorianus – “Lager yeast”, “bottom-

fermenting”• Product of interspecies hybridization

– Occasionally: Brettanomyces (some Belgian and craft beer styles), Torulaspora delbrueckii

Discovery of Saccharomyces eubayanus

Cytarria sp. - Beech gallsSugar-rich fruiting bodies

Lager yeast: a happy accident

Lager yeasts

• Lager represents >90% of global beer production

• Very limited diversity in yeasts available for these beer styles• Two lineages: Saaz and Frohberg• Very little genetic or flavour

diversity • But, now we know the other

parent!• Opportunity to develop new lager

yeasts with diversified flavour production

• Researchers performed spore-to-spore mating techniques using S. cerevisiae and S. eubayanus parents to produce diploid S. pastorianus yeasts

Breeding novel lager yeasts

Renaissanceyeast.com

Testing novel lager yeasts

Kristoffer Krogerus, Frederico Magalhães, Virve Vidgren, Brian Gibson. 35th Congress EBC Porto (2015)

“Alternative yeast” – Brettanomyces/Dekkera

• Brettanomyces/Dekkera and Saccharomyces diverged evolutionarily 250M years ago

• “Brett” usually a flaw in wines• Capable of desirable flavour production in beers• Recent trend with craft brewers• Some Brett strains can complete primary

fermentation – utilize maltose, maltotriose, etc• These fermentations are more fruity, less

“funky” or “medicinal” than wine equivalents • Some Brett strains have unique aroma characteristics – different from Saccharomyces

The wild side – sourdough bread and sour beer

• Both use mixed cultures of bacteria and yeast• Mutually beneficial

• Sourdough: • Bacteria break down starches and proteins in dough• Releases fermentable sugars and usable nitrogen for

yeast fermentation/nutrition• Yeast CO2/ethanol production inhibits growth of molds

and spoilage bacteria

The wild side – sourdough bread and sour beer

• Sour beer: • Traditional “Lambic” process – very starchy wort• Inoculated by wild microbes from air/brewery• Bacteria slowly break down starches and proteins,

causing slow release of nutrients and sugars for yeast• Slow fermentation (up to 1 yr, plus aging) results in

characteristic complex flavour