Brettanomyces Species = No Ascospore/Imperfect

VWT 272 Class 12 Quiz 8 Number of quizzes taken 12 Min 25 Max 32 Mean 29.4 Median 30 Mode 30 Lecture 12 Yeast and Yeast Nutrition • Kilgore Trout once wrote a short story which was a dialogue between two pieces of yeast. They were discussing the possible purposes of life as they ate sugar and suffocated in their own excrement. Because of their limited intelligence, they never came close to guessing that they were making champagne.

Kurt Vonnegut (1922-2007) Plan of Study

• Yeast Taxonomy • Yeast Species • Yeast Ecology • Yeast Nutrition

Yeast Taxonomy

• Yeast – Eukaryotic microorganism (has nucleus) – Fungus Kingdom (1% of all known fungi) – Evolved from a multicellular organism – Primarily reproduce asexually by budding – ~5 μm in length

Yeast Taxonomy • Classification of yeast is based on a form of reproduction – If, in response to “hard times”, the yeast makes Ascospores • If Ascospores formed = yeast is telomorph/sexual/perfect • If No Ascospores formed = yeast is anamoprph/asexual/imperfect • Examples – Metschnikowia pulcherrima = ascospore forming/perfect – Candida pulcherrima = no ascospore/imperfect

– Hanseniaspora uvarum = ascospore forming/perfect – Kloeckera apiculata = no ascospore/imperfect

– All Dekkera species = ascospore forming/perfect – All Brettanomyces species = no ascospore/imperfect

Yeast Taxonomy

– Silly and outdated • Now based on DNA

A Yeast Bestiary • Bestiary – a collection of pictures and stories about real of mythical animals. Popular in the Middle Ages (~ 500 CE to ~1400 CE) Candida • Candida krusei – Can co-ferment early in fermentation – Prefers low temperatures – Can contribute to ropiness – Used in making chocolate

– SO2 sensitive, EtOH to 6.6% • Candida stellata – Found in most must flora • More at the beginning than end – Prefers low temperatures – White, cheese like film on surface – Can produce honey/apricot characters

– SO2 somewhat sensitive, EtOH not sensitive • Candida vini – Uncommon spoilage yeast – Can “sour” or cloud wines

– SO2 sensitive > 50mg/L EtOH to 10%

Dekkera/Brettanomyces • Dekkera anomala/Brettanomyces anomalus – Can produce 4-ethylphenol & 4 ethylguaiacol • Barnyard, horse blanket, band aid, creosote – Can produce large amounts of acetic acid (VA) – Slow growing

– SO2 sensitive, EtOH to 10% • Dekkera bruxellensis/Brettanomyces bruxellensis – Can produce 4-ethylphenol & 4 ethylguaiacol – Usually observed during barrel ageing – Can spread through winery via contaminated equipment and insects – pH < 3.5 inhibit growth

– SO2 sensitive > 0.8 Molecular, EtOH >13%

Hanseniaspora/Kloeckera • Hanseniaspora guilliermondii/Kloeckera apis – Commonly found on damaged grapes – Sometimes dominant population in initial stage of uninoculated fermentations – Can “horde” thiamin – stuck fermentation

– SO2 slightly sensitive, EtOH > 5% • Hanseniaspora uvarum/Kloeckera appiculata – Often dominant population in initial stage of uninoculated fermentations • Usually outcompeted by S. Cerevisae by ~4% EtOH – Can produce ethyl acetate

– SO2 moderately sensitive, EtOH >9%

Metschnikowia • Metschnikowia pulcherrima/Candida pulcherrima – Commonly found on grapes & winery equipment • Resistant to most vineyard antifungal agents – Often large population in very early stage of uninoculated fermentations – Fast starter/short lag phase

– SO2 moderately sensitive, EtOH > 5%

Pichia • Pichia anomala/Candida pelliculosa – Commonly found on grapes – Active in initial stage of uninoculated fermentations – Can form film on top of wine – Can form lots of acetic acid/ethyl acetate

– SO2 sensitive, EtOH slight sensitivity • Pichia guilliermondii/Candida guilliermondii – Can form cheese-like film on top of wine – Can produce ethyl acetate

– SO2 moderately sensitive, EtOH >9% • Pichia Subpelliculosa/Hansenula subpelliculosa – Commonly found on grapes – Weak fermenter – Common pathogen on strawberries

– SO2 tolerant, EtOH tolerant

Saccharomyces • Saccharomyces bayanus – Extremely common commercial wine yeast – Commonly found in wineries – “Champagne” type yeast – Can “horde” nutrients – Very strong fermenter

– SO2 tolerant, EtOH highly tolerant • Saccharomyces cerevisiae – Most common commercial wine yeast • Most important commercial microbe – Commonly found in wineries – Very strong fermenter

– SO2 tolerant, EtOH tolerant

Schizosaccharomyces • Schizosaccharomyces pombe – Can use Malic Acid as energy source, so can reduce acidity in wine – Reproduces by simple fission – Can produce unpleasant S compounds

– SO2 moderately sensitive, EtOH moderately sensitive

Zygosaccharomyces • Zygosaccharomyces bailii – Extremely tolerant to high sugar concentration • Can grow in grape juice concentrate – Extremely tolerant to high alcohol concentration • Can grow in wines > 18% Ethanol

– Tolerant to high SO2 concentrations – Can cause refermentation in sweet wines – Very slow growth – Can cause haze in wine

Welcome to the (Grape) Jungle! • On grape surface estimated between 104 and 105 yeast per square cm • Over 50 species of yeast have been identified – Saccharomyces cerevisiae is very rare! • Yeast type changes by ripeness, and location on the berry • Insects are vectors of yeast dispersal in vineyards Welcome to the (Winery) Jungle! • Following grape processing Saccharomyces populations can increase < 3 orders of magnitude – 10 to 10,000 cells/mL • Saccharomyces can produce biofilms – Common with Saccharomyces, Brettanomyces, and Candida yeast • Behavior increased with presence of polyphenols • Many winery surfaces are very difficult to clean – Wood – Concrete Welcome to the (Fermentation) Jungle! • In uninnoculated fermentations rely on grape and winery based yeast • Early fermentation are dominated by non-Saccharomyces yeast – Temperature of fermentation determines which non- Saccharomyces yeast will dominate • Hanseniaspora and Candida species more dominant in cold ferments • Mid fermentation is characterized by the increase of population of Saccharomyces yeast • Late fermentation are highly dominated by Saccharomyces yeast • In innoculated fermentations Saccharomyces yeast quickly dominate population Welcome to the (Aging) Jungle! • Type of organisms present – Winery practices – Sanitation – Storage vessels • Wood & Cement – hard to clean • Stainless Steel – easier but not perfect – Yeast found in non-biologically active state • Candida, Pichia, Brettanomyces & Zygosaccharomyces

Yeast Growth Curve

Number of of Numbercells

Time Yeast Nutrition • Energy – From Glucose/Fructose

• Micronutrients – From grape juice or supplement – No commercial analysis for quantification – Biotin, pantothenate, potassium, sterols, fatty acids • Nitrogen source – Free Amino acids (FAN) • Not peptides or proteins • Not Proline + – Ammonium salts (NH4 ) + • FAN + (NH4 ) = Yeast Assimilable Nitrogen (YAN) Yeast Nutrition • YAN highly variable – Dependant on • vineyard nutrition • Rootstock + • FAN + (NH4 ) = Yeast Assimilable Nitrogen (YAN)

n=1524 Free Amino N Ammonium YAN mg N/L mg N/L mg N/L Mean 135 79 213 Min 29 5 40 Max 370 325 559 + • NO relationship between FAN and NH4 concentrations Yeast Nutrition • FAN – Determined in a lab – NOPA – Report as mg N/L + • NH4 – Determined in a lab – Ion selective electrode – Report as mg N/L + • YAN = FAN + NH4 + • NO relationship between FAN & NH4

YAN Requirements

• How Much? Total YAN (in grape + additive) ≤ 23 °Brix 250 mg N/L > 23 but < 25 °Brix 300 mg N/L ≥ 25 °Brix 350 mg N/L (or more) • Can there be too much N? – Excess N leads to • High Fermentation Rate – High temperature fermentations – Fast/Hot fermentations = less aroma complexity & amount • Leftover N for spoilage organisms YAN Requirements • When to add? – Professor Linda Bisson (UC Davis) • After Saccharomyces have become dominant – “No reason to feed the yeast you don’t want!” • Ethanol is inhibitory to N transport into the yeast cell • Some nutrients needed for alcohol tolerance need to be available when new cells are being made Feed 24 to 48 hours after inoculation – Lisa van der Water (BSG Wine Division) • At yeast rehydration – Gives yeast a boost for a fast start • At yeast inoculation • During the first half of fermentation – Multiple additions – After yeast have stopped growing but before they are incapable of uptake Feed early and often

YAN Requirements • What to add? • Diammonium phosphate DAP + – Provides NH4 – And phosphate (also used by the yeast) – Cheap • Yeast autolysate – Provides amino acids – Provides micronutrients – Vary widely in quality – Expensive • “Blends” of DAP and Autolyzed yeast – Invented by Lisa van der Water (Superfood)

Yeast Inoculation 5/10 Method 1. Prepare a chlorine-free, 5:1 water to juice mixture of approximately 5 times the yeast weight at 35 - 40°C (95 - 104°F). 2. Gently sprinkle the yeast into the water-juice mixture and, without stirring, allow 10 minutes for hydration. 3. Stir the yeast into the water-juice mixture allowing it an additional 10 minutes to fully hydrate. 4. Add enough juice to the yeast suspension to lower the temperature by 5°C. Let mixture acclimate for 5 minutes. 5. Repeat above until the temperature of the yeast suspension is less than 10°C warmer than the must or juice to be inoculated. 6. Slowly add the yeast mixture into one area of the must or juice. Do not mix or pumpover for at least 10 hours Next Week

• Bacteria – The GOOD – The BAD – The UGLY