Optimization*of*Non-Saccharomyces Yeasts*for*Production*of*Lower*Ethanol*Red*WinesTara*Cook1,*Victoria*Paup1,*Curtis*Merrick1,*Carolyn*F.*Ross1 and*Charles*G.*Edwards1

1School*of*Food*Science,*Washington*State*University,*Pullman,*WA

ACKNOWLEDGEMENTSThe$authors$thank$the$Washington$State$Grape$&$Wine$Research$Program,$Ste.$Michelle$Wine$Estates,$and$the$School$of$Food$Science$for$financial$and$material$support.

REFERENCESAplin,$J.J.,$Cook,$T.L.,$Edwards,$C.G.$Evaluation$of$nonFSaccharomyces yeasts$isolated$from$Washington$vineyards$to$reduce$final$alcohol$contents$

of$Merlot$wines.$Am.$J.$Enol.$Vitic.$(Submitted$2020).Aplin,$J.J.,$White,$K.P.,$Edwards,$C.G.,$2019.$Growth$and$metabolism$of$nonFSaccharomyces yeasts$isolated$from$Washington$state$vineyards$in$

media$and$high$sugar$grape$musts.$Food$Microbiol.$77,$158–165.Baker,$A.K.,$Castura,$J.C.,$Ross,$C.F.,$2016.$Temporal$CheckFAllFThatFApply$Characterization$of$Syrah$Wine.$J.$Food$Sci.$81,$1521–1529.$Contreras,$A.,$Hidalgo,$C.,$Henschke,$P.A.,$Chambers,$P.J.,$Curtin,$C.,$Varela,$C.,$2014.$Evaluation$of$nonFSaccharomyces$yeasts for$the$reduction$

of$alcohol$content$in$wine.$Appl.$Environ.$Microbiol.$80,$1670–1678.$De$Deken,$R.H.,$1966.$The$Crabtree$effect:$A$regulatory$system$in$yeast.$J.$Gen.$Microbiol.$44,$149–156.$Erten,$H.,$2002.$Relations$between$elevated$temperatures$and$fermentation$behaviour$of$Kloeckera.apiculata.and$Saccharomyces.cerevisiae.

associated$with$winemaking$in$mixed$cultures.$World$J.$Microbiol.$Biotechnol.$18,$377–382.Gao,$C.,$Fleet,$G.H.,$1988.$The$effects$of$temperature$and$pH$on$the$ethanol$tolerance$of$the$wine$yeasts,$Saccharomyces.cerevisiae,.Candida.

stellata and$Kloeckera.apiculata.$J.$Appl.$Bacteriol.$65,$405–409.Godden,$P.,$Wilkes,$E.,$Johnson,$D.,$2015.$Trends$in$the$composition$of$Australian$wine$1984F2014.$Aust.$J.$Grape$Wine$Res.$21,$741–753.$Goldner,$M.C.,$Zamora,$M.C.,$Lira,$P.D.L.,$Gianninoto,$H.,$Bandoni,$A.,$2009.$Effect$of$ethanol$level$in$the$perception$of$aroma attributes$and$the$

detection$of$volatile$compounds$in$red$wine.$J.$Sens.$Stud.$24,$243–257.Gonzalez,$R.,$Quirós,$M.,$Morales,$P.,$2013.$Yeast$respiration$of$sugars$by$nonFSaccharomyces yeast$species:$A$promising$and$barely$explored$

approach$to$lowering$alcohol$content$of$wines.$Trends$Food$Sci.$Technol.$29,$55–61.$King,$E.S.,$Dunn,$R.L.,$Heymann,$H.,$2013.$The$influence$of$alcohol$on$the$sensory$perception$of$red$wines.$Food$Qual.$Prefer.$28,$235–243.$Mira$de$Orduña,$R.,$2010.$Climate$change$associated$effects$on$grape$and$wine$quality$and$production.$Food$Res.$Int.$43,$1844–1855.Villamor,$R.R.,$Ross,$C.F.,$2013.$Wine$matrix$compounds$affect$perception$of$wine$aromas.$Annu.$Rev.$Food$Sci.$Technol.$4,$1–20.

RESULTS/DISCUSSIONThe$most$important$finding$in$this$work$was$the$use$of$nonFSaccharomyces

yeasts$resulted$in$significant$reductions$of$ethanol$in$largeFscale$(120kg)$fermentations$(Table$1).$This$was$a$result$of$temperature$optimization,$which$extended$nonFSaccharomyces survival$during$fermentation,$and$thus$increased$fermentable$sugars$utilized$by$these$yeasts.$Total$nonFSaccharomyces yeast$populations$quickly$declined$in$fermentations$inoculated$with$S..cerevisiae.alone$(undetectable$by$day$9),$while$fermentations$inoculated$with$Mt..pulcherrima and$My..guilliermondii displayed$nonFSaccharomyces$populations$until$day$13$and$19$(Figure$1).$An$increase$in$ethanol$tolerance$of$nonFSaccharomyces yeasts$at$low$temperatures$has$been$previously$reported$(Erten,$2002;$Gao$and$Fleet,$1988).$

Previous$work$in$our$laboratory$has$shown$the$use$of.Mt..pulcherrima.and$My..guilliermondii.do$not$lead$to$reductions$in$wine$quality$(Aplin$et$al.,$2019,$2020).$This$was$also$observed$in$the$present$study$as$sensory$panel$data$revealed$that$wines$with$the$highest$ethanol$reduction$(Mt..pulcherrimafollowing$temperature$regime$B)$were$lower$in$attributes$of$‘bitter’$(taste),$‘ethanol’$(flavor),$and$‘hot/ethanol’$(mouthfeel)$as$shown$in$Figure$2.$

ABSTRACTViticultural$systems$that$yield$‘rich’,$‘fullFbodied’$red$wines$often$involve$

extended$vine$ripening$periods$to$achieve$phenolic$maturity.$However,$these$practices$result$in$increased$concentrations$of$sugar$which$subsequently$leads$to$higher$amounts$of$ethanol.$As$a$means$to$reduce$the$ethanol$contents$of$wines,$selected$nonFSaccharomyces yeasts$have$been$studied$to$metabolize$glucose$and$fructose$in$grape$must$prior$to$initiation$of$alcoholic$fermentation$by$S..cerevisiae.$However,$little$work$has$been$performed$to$optimize$fermentation$temperatures$to$encourage$these$nonFSaccharomyces yeasts.$In$2018,$Merlot$grapes$were$crushed/destemmed$with$120$kg$distributed$into$temperature$controlled$300$L$fermentation$tanks.$Musts$were$then$inoculated$with$either$Metschnikiowa.pulcherrima or$Meyerozyma.guilliermondii at$106CFU/mL$prior$to$adding$S..cerevisiae D254.$While$control$tanks,$inoculated$with$S..cerevisiae alone,$were$set$to$a$maximum$of$25°C,$treatments$were$set$to$a$maximum$of$either$15°C$(temperature$regime$A)$or$17.5°C$(temperature$regime$B)$before$adding$S..cerevisiae.on$day$3.$At$this$time,$temperature$maximums$were$changed$to$25°C.$Once$fermentations$achieved$dryness$(<2$g/L$residual$sugar),$wines$were$bottled$and$stored$for$6$months$at$7°C$prior$to$sensory$analysis.$The$inoculation$of$nonFSaccharomyces yeasts$into$initially$lower$temperature$musts$was$effective$in$extending$culturability$of$nonFSaccharomyces yeasts$during$fermentation$and$led$to$reductions$in$final$ethanol$contents.$Here,$wines$inoculated$with$Mt..pulcherrima.following$temperature$regime$B$prior$to$inoculation$of$S..cerevisiae.achieved$the$greatest$reduction$in$ethanol,$approximately$0.7%$v/v.$Additionally,$panelists$found$this$treatment$to$express$lower$sensory$scores$for$‘hotness’,$‘bitterness’,$and$‘ethanol$flavor’.$In$agreement$with$past$studies$conducted$by$our$laboratory,$inoculation$of$either$Mt..pulcherrima.or$My..guilliermondii.into$musts$followed$by$S..cerevisiae.led$to$reductions$in$alcohol$contents$without$reductions$in$quality.

INTRODUCTIONAverage$alcohol$concentrations$of$wines$have$risen$due$to$extended$vine$

hangFtime$to$achieve$phenolic$maturity$and$thus$higher$sugar$concentrations$at$harvest$(Godden$et$al.,$2015;$Goldner$et$al.,$2009).$Increases$in$alcohol$content$can$negatively$affect$wine$quality$and$sensory$attributes.$With$increases$in$ethanol,$attributes$of$bitterness,$astringency,$and$ethanol$burn$tend$to$be$intensified$(Baker$et$al.,$2016;$Villamor$and$Ross,$2013)$while$fruity$attributes$tend$to$be$masked$(Goldner$et$al.,$2009;$King$et$al.,$2013).$In$addition,$higher$alcohol$wines$risk$rejection$by$health$conscious$consumers$and$may$incur$additional$taxation$(Gonzalez$et$al.,$2013;$Mira$de$Orduña,$2010).

Gonzalez$et$al.$(2013)$suggested$the$use$of$nonFSaccharomyces yeasts$in$conjunction$with$S..cerevisiae for$ethanol$reduction.$The$Crabtree$effect$is$not$observed$in$many$nonFSaccharomyces species$(De$Deken,$1966);$therefore,$these$yeasts$would$partially$consume$fermentable$sugars$to$metabolites$other$than$ethanol.$While$ethanol$reductions$of$up$to$1.6$%$(v/v)$have$been$reported$in$small$scale$laboratory$fermentations$(Contreras$et$al.,$2014),$few$have$reported$the$largeFscale$use$of$nonFSaccharomyces yeasts.$Additionally,$optimization$of$fermentation$temperature$for$ethanol$reduction$has$not$been$reported.$Previous$work$in$our$laboratory$has$shown$the$potential$for$ethanol$reduction$without$negatively$influencing$wine$quality$using$selected$nonFSaccharomyces.strains$(Aplin$et$al.,$2020,$2019).$The$aim$of$this$work$was$to$optimize$fermentation$temperature$for$use$of$selected$nonFSaccharomycesyeasts$to$reduce$final$ethanol$concentration$in$red$wines$without$affecting$wine$quality.$

EXPERIMENTAL*DESIGN Table*1.*Composition$of$Merlot$wines$inoculated$with$S..cerevisiae.and$fermented$at$25°C$or$with$selected$nonFSaccharomyces yeasts$following$temperature$regime$A$or$B$prior$to$inoculation$of$S..cerevisiae on$day$3

aFc$Mean$values$within$columns$with$different$superscripts$are$significantly$different$(p≤0.05)$$nd:$not$detected$(below$limit$of$detection$<0.07$g/L$)

Merlot$Grape$Must$(145$g/L$glucose,$142$g/L$fructose)120$kg$must$per$fermenter

Control Temperature*Regime*A

Temperature*Regime*B

Set$to$maximum$

25°C

Set$to$maximum$15°C$for$3$days,$then$25°C$for$

remainder

Set$to$maximum$17.5°C$for$3$days,$then$25°C$for$

remainder

! Inoculated$in$duplicate$with$Metschnikiowa.pulcherrima.P01A016$orMeyerozyma.guilliermondii P40D002$at$106 CFU/mL$on$day$0

! Inoculated$with$Saccharomyces.cerevisiae.D254$on$day$3

! Inoculated$in$duplicate$with$Saccharomyces.cerevisiae D254$on$day$0

Wines$sterile$filtered$(0.45$μm)750$mL$nitrogen$flushed$bottles$filled

Stored$6$months$at$7°C

Trained$Sensory$Analysis$Panel High$Performance$Liquid$Chromatography

• 10$Panelists• 12$hours$of$training• 44$total$aroma,$taste,$

mouthfeel,$and$flavor$attributes$assessed

• Wines$evaluated$in$triplicate

• Glucose• Fructose• Organic$

Acids• Ethanol

RESULTS

Figure*1.*Culturable$populations$of$S..cerevisiae.($$$),$total$nonFSaccharomyces yeasts$($$$),$and$temperatures$($$)$of$Merlot$fermentations$inoculated$on$day$0$with$S..cerevisiae.and$set$to$25°C$(A),$or$with$Mt..pulcherrima.(B,D)$or$My..guilliermondii.(C,E)$$following$temperature$regime$A$(B,C)$or$temperature$regime$B$(D,E)$until$sequentially$inoculated$with$S..cerevisiae.on$day$3.$*Asterisk$denotes$wines$pressed$at$0°Brix$and$kept$at$21°C.$

< 600

103

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109

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*

0 4 8 12 16 20 24

Time (days)

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< 600

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< 600

103

105

107

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Popu

latio

ns (C

FU/m

L)Tem

perature (°C)

0

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*

0 4 8 12 16 20 240

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A

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D E

Figure*2.*Spider$chart$of$mean$values$for$attributes$found$to$be$significant$(*p≤0.05,$**p≤0.01,$***p≤0.001)$by$trained$panel$(n=10)$evaluation$of$2018$Merlot$wines$inoculated$with S..cerevisiae and$fermented$at$25°C$or$with$selected$nonFSaccharomyces yeasts$following$temperature$regime$A$or$B$prior$to$inoculation$of$S..cerevisiae on$day$3.$+Denotes$replicates$could$not$be$pooled.$

5.5

6.5

7.5A-Dried Fruit*

A-Sulfur*

A-Woody*

A-Herbaceous*

A-Spicy***

A-Yeasty**

T-Sweet*

T-Bitter**

T-Sour**

M-Tingle**

M-Hot/Ethanol**

M-Roughness***

M-Astringent**M-Drying***M-Puckering***

M-Sharp***

M-Mouthcoat*

M-Round**

F-Berry*

F-Sweaty*

F-Dried Fruit**

F-Ethanol***

F-Sulfur**

F-Solvent*

F-Animal*

F-Herbaceous**

My. guilliermondii A-1+

S. cerevisiae

My. guilliermondii BMy. guilliermondii A-2+Mt. pulcherrima A Mt. pulcherrima B

3.5

4.5

FUTURE*RESEARCHFermentations$from$the$same$Merlot$vineyard$block$have$been$conducted$in$

fall$of$2019.$Optimization$for$timing$of$sequential$inoculation$of$S..cerevisiaewith$Mt..pulcherrima.was$studied.$Wines$are$currently$undergoing$analysis.$