Food Biotechnology: Alcohol and Beer

Food Biotechnology

  • Lecturers: Dietmar Haltrich, Clemens Peterbauer

  • Department of Food Sciences and Technology

Alcohol

  • Sugar converted to ethanol by yeasts.

  • Ethanol concentration limited by sugar, yeast tolerance (around 15% v/v).

  • Distillation increases ethanol concentration.

  • Fortified wines: addition of partially purified ethanol.

Alcoholic Beverages

  • Ethanol mainly from Saccharomyces spp.

  • Zymomonas mobilis in African palm wines.

  • Key trait: fermenting sugars into ethanol.

  • Crabtree-positive yeasts use fermentation even with oxygen.

The Crabtree Effect

  • Yeast produces ethanol aerobically at high glucose, instead of biomass via the TCA cycle.

  • Observed in many genera.

  • High glucose accelerates glycolysis, reducing need for oxidative phosphorylation.

  • Believed to be a competition mechanism, ethanol's antiseptic nature.

ATP Production in Yeasts

  • 2 ATP per glucose via fermentation.

  • ~18 ATP per glucose for yeast with low P/O ratio in oxidative phosphorylation.

Evolution of the Crabtree Effect

  • Progenitor was strictly aerobic.

  • Saccharomyces underwent whole genome duplication (WGD) ~100 million years ago.

  • WGD increased flux in glycolysis.

  • Duplication of alcohol dehydrogenase: Adh1 (ethanol formation), Adh2 (ethanol uptake).

  • 'Make-accumulate-consume' strategy: yeasts ferment glucose to defend resources, consume ethanol later.

Alternative Explanation: Rate/Yield Trade-off Hypothesis

  • Limited respiration capacity.

  • Excess sugar processed via fermentation.

  • Faster ATP production rate, lower yield.

  • Rate more relevant for competitive fitness.

BEER

Beer - History

  • Originated in Mesopotamia ~8000 years ago.

  • Used to pay for labor in Mesopotamia and Egypt.

  • Germanic and Celtic tribes produced beer ~1000 years ago.

  • Greeks/Romans valued wine more.

  • Flavor from plants like sweet gale (Myrica gale).

Brewing Process Control

  • Controlled in monasteries, gruit replaced by hops.

  • 'Reinheitsgebot von 1516': water, barley, hops only.

  • Hops for flavor and preservation.

  • European style beer is now most popular.

World Beer Production

  • Top producers: China, United States, Brazil, Germany, Russia, Mexico, Japan, United Kingdom, Poland, Spain

Beer Production Specifics

  • Principal cereal: barley.

  • Africa: sorghum, millet with lactic/ethanolic fermentation.

  • Low gelatinization temperature of malted barley starch (52-59°C).

  • β-amylase: rapid starch conversion to maltose.

  • Saccharomyces cerevisiae can't directly ferment starch.

  • Starch conversion: mould enzyme (Oriental), endogenous enzymes (Western), salivary amylase (South America).

Malting

  • Germination under control.

  • Green malt -> kiln-dried malt.

  • Formation of enzymes, breakdown of cell walls.

  • Hydrolytic enzymes mobilize nutrients.

  • Gibberellins added.

  • Mostly two-rowed barley, low protein.

Steeping

  • Dried barley (<12% water).

  • Activity increases at >30% water, rapid >38%, optimal enzyme formation >43%.

  • Steeping in water with air rest, 24-48 h total.

Germination

  • Optimal: 43-40% water.

  • 14-18 °C.

  • Oxygen needed; heat produced.

  • Rootlet length indicates process.

Enzymes Formed During Malting

  • β-glucanases, Pentosanases, Proteases, Amylases, Lipases

Malting Processes

  • Floor malting (Tennenmälzerei): 10-cm barley layer, T controlled by mixing, 6-8 days (light malts), 8-11 days (dark malts).

  • Pneumatic germination plants: humid air cools grains, Saladin box (1-m layer), 7 days, T increases from 12° to 18°C.

Kilning

  • Reduce water: >40% to 1.5-2% (dark) or 3.5-4% (light).

  • Rootlets removed.

  • Further growth up to 40°C, water removal at lower T to ~10% water.

  • Enzymes active/stable at 40-70°C & >10% water.

  • Final kilning: light malt >80 °C, dark malt 100-105°C.

  • 100 kg barley -> 160 kg green malt -> ~80 kg kiln-dried malt.

Maillard Reaction

  • T > 100°C, amino acids + sugars = color and flavor.

  • Amino group + carbonyl group -> N-substituted glycosylamine -> Schiff base -> N-substituted amino-deoxyketone -> melanoids.

Sulfur Compounds

  • Formed during kilning, e.g., dimethyl sulfide (DMS).

  • DMS from S-methylmethionine (SMM), evaporates at higher T.

  • Beer concentration: 10-150 µg/liter.

Malts

  • Lager/Pils, Pale Ale, Carapils, Crystal, Chocolate, Black

Special Malts

  • Wheat, rye, triticale malts.

  • ‘Farbmalz’, ‘Caramelmalz’, ‘Brühmalz’

Braumalz Stadlauer Malzfabrik

  • Pilsner, Wiener, Münchner, Karamellmalz extrahell, Karamellmalz dunkel, Weizenmalz, Farbmalz

From Malt to Wort

  • Malt ground, mixed with hot water.

  • Water quality matters; calcium ions precipitate, lowers pH.

  • British infusion: single vessel, 65°C.

  • Continental decoction: heated through temperatures.

Steps

  • Grind malt, mash, boil -> wort (fermentable sugars).

  • Separate extract from insolubles (lautering/filtration).

  • Boil wort with hops.

  • Cool wort.

Raw Materials for Wort

  • Malted barley.

  • Wheat malt in top-fermented beers.

  • Unmalted cereal (barley, rice, maize).

  • Limit: 30% in Europe, 50% in USA.

  • Milling: grind contents, crack husk.

Mashing

  • Steep grist in warm water, different temperatures.

  • Unmalted cereal heated to gelatinization T.

  • Enzymatic starch degradation: maltose (40-45%), maltotriose (11-13%), glucose (5-7%), dextrins.

  • Degradation of cell walls and protein.

Carbohydrate Composition of Wort (in %)

  • Fructose, glucose, sucrose, maltose, maltotriose, maltotetrose, total dextrins, total fermentable sugars

Continental Decoction Mashing

  • Step-wise T increase.

  • Mash heated at 100 °C, mashing at 35-37°C.

  • Next mash at 50-53°C: protein hydrolysis, starch degradation.

  • Next mash at 62-67°C: starch degradation.

  • 5-6 h total; Maillard reactions.

Infusion Mashing

  • British ales, Belgium, northern Germany.

  • Wort drawn off, replaced by hot sparge water.

  • Originally 70°C; now heated in steps.

  • Filtered in lauter tun or mash filter.

Wort Separation (Lautering – Läuterung)

  • Lauter tun: horizontally slotted metal base.

  • Mash filter: filter press, cereal solids form filter aid, wort squeezed out.

Boiling and Hops Addition

  • Concentrate wort, desired strength.

  • Inactivate enzymes, precipitate proteins.

  • Sterilization.

  • Solubilize hops, isomerize hop acids.

  • 1–2 h duration.

  • Remove undesired flavor components (DMS).

Hops

  • Function: bitter taste, flavor, conservation, protein precipitation.

  • Varieties: Hallertau, Saaz, USA (Cluster, Galena).

  • Main components: Resins (α, β-acids), tannins, essential oils.

Hop Acids

  • α-acids (humulone), β-acids (lupulone)

  • see table for composition

Importance of α-acids

  • More important for bitterness, low solubility (solubilized by boiling).

  • Isomerization to iso-α-acids during boiling, improved solubility.

  • Use cone hops, pellets, or hop extract.

  • Pre-isomerized products added before cooling.

Fermentation

  • Bottom-fermenting yeasts, Saccharomyces pastorianus (S. cerevisiae x S. eubayanus).

  • From 1860 on in Central Europe.

  • Saaz-type and Frohberg-type lager yeasts.

  • S. eubayanus closely related to S. cerevisiae.

Origin Models for Saaz and Frohberg

  • Hybridization events between distinct diploid S. cerevisiae and diploid S. eubayanus parental strains.

More on Lager Yeast

  • 5-10 °C fermentation.

  • Flocculation, sediments.

  • minor differences between them (e.g., formation of esters, etc.).

More on Ale Yeast

  • Top-fermenting yeasts, Saccharomyces cerevisiae.

  • 15-25° C fermentation.

  • hydrophobic surface causes the flocs to adhere to CO2CO2​

  • Far more diversity among ale strains.

Yeast Flocculation

  • Reversible, asexual, calcium-dependent.

  • Cells adhere to form flocs.

  • Separate from medium by sedimentation (lager) or rising (ale).

  • Lectin-like proteins (flocculins) bind to cell-wall mannose, requires Ca2+Ca2+.

Beer Yeast Phylogeny

  • Beer 1, Beer 2, and Mixed lineages.

  • Genetically distinct, long evolution in rich medium.

Beer Yeast Domestication

  • Separation of lineage Beer 1 and 2 around 1600–1700.

  • Geographic pattern within lineages.

  • Accentuation of desirable traits.

  • Effective maltotriose utilization, reduced 4-vinyl guaiacol production.

  • Ethanol production typically 7.5–10%.

Maltotriose Utilization

  • Important sugar in wort.

  • MAL11 transporter, high affinity for maltotriose, in beer yeasts.

4-Vinyl Guaiacol

  • From ferulic acid, off-flavour (spicy, clove-like).

  • Detoxification reaction.

Ferulic Acid Decarboxylase

  • Most beer yeasts lost ability, but some wheat beer yeasts still form it.

Non-Saccharomyces Yeasts - Brettanomyces

  • In certain beer types, cask-aged beers.

  • Brettanomyces claussenii.

  • First patent on a microorganism!

  • Brettanomyces (anamorph), Dekkera (teleomorph).

  • Spoilage in wine, valued in beer.

  • B. bruxellensis common.

Brettanomyces vs. Saccharomyces

  • B. bruxellensis and S. cerevisiae separated ~200 million years ago.

  • High resistance to osmotic, ethanol stress.

  • Growth in oxygen-limited, low pH.

  • Crabtree-positive.

Brettanomyces spp.

  • Optimum growth ~25° C.

  • Crabtree-positive, Custers effect.

  • Ferments glucose, maltose, higher maltodextrins.

  • Forms acetate aerobically.

Comparison of Brettanomyces, Saccharomyces pastorianus, and Saccharomyces cerevisiae

  • Summary of brewing features.

More on Brettanomyces in Beer

  • Special beers (lambic, Belgian sour beers, Kriek, geuze).

  • Second fermentation of cask matured beers.

  • Produces esters, 4-ethylguaiacol, 4-ethylphenol.

Use of Brettanomyces in Lambic-style Beers

  • Long fermentation/maturation (1-3 years).

  • Sugars by Saccharomyces, then Brettanomyces after 4-8 months.

  • Lactic acid bacteria (Lactobacillus, Pediococcus) active.

Starter Cultures

  • Reuse yeast (up to 15 times).

  • Pitching yeast, aeration.

  • Yeast population increases ≈8x.

  • 12° wort fermentation: 6 to10 days.

  • Cylindroconical vessels, up to 6,000 hl.

Conditions and Processes

  • Lager fermentation: 8 – 12°C

  • Ale fermentation: 12 – 18°C

  • Carbohydrates -> ethanol (EMP pathway).

  • Anaerobic, aeration for fatty acid biosynthesis.

  • Vigorous fermentation, then slower.

  • ‘Green beer’ contains by-products.

Conditioning of Beer

  • Lager: storage at 0 to 2°C.

  • Cask-conditioned: priming sugar for secondary fermentation.

Lagering By-Products

  • Acetaldehyde, diacetyl, esters, higher alcohols, yeast concentration, total extract , and phenolic compounds are some of the key by-products that influence the flavor and aroma profile of the beer during the lagering process.

Beer Flavour

  • Bitterness: 5-50 mg/L iso-α-acids, rather labile and decompose in light and with oxygen.

  • Esters: produced by yeasts; ethyl acetate (20-40 mg/L), isoamyl acetate (2-5 mg/L).

  • Aldehydes (from oxidation of alcohols or from lipids), acetaldehyde, 5-10 mg/L.

  • Higher alcohols: 2-methylpropanol, 2-methylbutanol, 3-methylbutanol, 2-phenylethanol (from yeasts).

  • 4-Vinylguaiacol: phenolic or clove-like flavor, from ferulic acid through thermal or enzymatic decarboxylation.

  • Vicinal diketones: 2,3-butanedione (diacetyl), 2,3-pentanedione, formed from α-acetolactate, degraded by yeast; the threshold value of diacetyl in Pilsner beer is 0.03 mg/L.

  • Sulfur components: dimethyl sulfide (DMS).

Beer Flavour Components

  • A diagram outlining the various organic acids, sugars, amino acids, vicinal diketones, aldehydes, ethanol, fatty acids, lipids, and esters that contribute to beer flavor, alongside the metabolic pathways that link them.

Organic Acid Concentrations

  • A table listing major organic acids found in beer and their typical concentration ranges (mg/L):

    • Acetic acid: 30-200

    • Propanoic acid: 1-5

    • Butanoic