ma lo lactic fermentation

8/6/2019 Ma Lo Lactic Fermentation

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The Joy ofMalolactic Fermentation

Ellen ButzDept. of Food Science, Purdue University

Phone: [email protected]


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Malolactic FermentationThe Second Fermentation1913 Mller-Thurgau proved bacterialtheoryC4 H6 O5 C3 H6 03 + C O 2

MALIC ACID LACTIC ACID CARBON DIOXIDE

Lactic Acid Bacteria LAB- 4 genera:

LactobacillusPediococcusLeuconostocOenococcus


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Malolactic Fermentation

Oenococcus/Leuconostoc LactobacillusPediococcus

Schizosaccharomyces pombe

Saccharomyces cerevisiaeGenetically modified with bacterial DNA


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All LABGram PositiveNon-mobile & non-sporulatingFacultative AnaerobesConvert Malic Acid to Lactic AcidRequire rich medium & fermentable

sugarsDifferent genera utilize glucose differently

Taxonomy of LAB


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Desired character of final wine?

Acid level reductionDepends on concentration of Malic AcidRough estimate with paper chromatography

Masking of vegetal character by diacetylIncreased body by increased production ofdextrans and glucans

Increased flavor complexityButtery, nutty, honey vanilla, leather, spicestoasty, more body, smoother tannins

MLF Decisions Why to


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To encourage high levels:MLF occurs late in fermentationLower MLF culture inoculation rateLower end of temperature rangeLower pH winesHigh Citric Acid concentrationReduce contact time with yeast andbacterial lees

Diacetyl ConcentrationThe Butter Factor


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Diacetyl ConcentrationThe Butter Factor

malate

citrate

lactate

acetate

biomasssugars (fructose)

CELL GROWTHsugar catabolism

no malate catabolismno citrate catabolismslight acetate productionslight lactate production

STATIONARY PHASE Ino sugar catabolism

malate catabolismno citrate catabolismno acetate productionlactate production

STATIONARY PHASE IIno sugar catabolism

no malate catabolismcitrate catabolismacetate productionno lactate production


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Potential problems:

Volatile acidity always increases,especially in high pH winespH is always increasedCost to produce wine increasedTime to produce wine increasedSulfur dioxide addition delayedNOT recommended for sweet wines

MLF Decisions Why not


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Spoilage issues involveWine conditions above 3.5 pH &

Indigenous Lactobacillus andPediococcus (not Oenococcus)Off odors:

Cheesy, milky, metallic, earthy, mousy,sweaty, wet leather, rotten fruitPhysical changes:

Loss of colorRopinessBitterness

Tartaric acid decomposition

Wine Spoilage by LAB


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Spoilage issues involveProduction of Histamine

May cause rx at levels >0.1 mg/L insensitive people

Rashes, respiratory problems, runny noseHi pH >3.5 = increased Histamine levelsInoculation with commercial cultures

reduces population of wild LABPost MLF cells should be removed ASAPto reduce histamine production

Wine Spoilage by LAB


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Wine Conditions vs MLF

Late yeastfermentation

Aged, clarifiedwine

Inoculation

20 mg/LFree SO2

30 mg/LTotal SO2

13.5%Alcohol

>3.2


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Wine Conditions vs MLF


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Inoculation near end of sugar fermentation

usually bestWarmer temperatures (60-65 F)Lower CO2 concentrationDeteriorating yeast cells

contribute micronutrientsAbsorb inhibitory compounds

pesticides, decanoic acidHigher concentration of flavor compounds

Diacetyl, acetoin & 2,3 butanediol

MLF Decisions Timing


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Uninoculated/Spontaneous MLF

Why? you forgot to buy MLF culture you like to take big chances it just happened flavor complexity potential increased body


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Uninoculated/Spontaneous MLF

How?

Hi pH wines, used barrels, warmtemperatures, red winesMinimal SO2 addition


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Using Starter Cultures

4-8% of final winevolume (pH>3.2, 65-80 oF).Wait for ~1/2 conversionL-malic before expanding

(1-3 weeks)

1 rehydrated sachet(68-86 oF clean

water)slight stirring, avoidair, let suspension

rest 15 minutes

Equal parts H 2O & juice (pH>3.3, noSO2), 1% of final

wine volume, addactive dried yeast &

ML nutrient. Wait formix to complete

alcohol fermentation.(3-5 days)

DIRECT INOCULATION CULTURES

BUILD-UP CULTURES

1.

2.


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Problem ML Fermentations

Must monitor with chromatography or better

evidence of gas or reduced totalacidity is not good enoughConsider

Warming slightly if lower than optimumwine temperature

Racking or stirring to resuspend packedleesReinoculation (after 3-4 weeks)


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Alternative MicrobesSchizosaccharomyces pombe ProMalic

Considered spoilage yeast can product offodors during late alcoholic fermentationMetabolizes malic acid to ethanol no lactic

acid producedEncapsulated productremove when malic

acid is depleted


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ProMalic


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ProMalicPros & Cons

Pros:

Reliable, fast, easy, no lactic acid,more acid reduction, tolerates SO2 &very low pH

Cons:Expensive, cannot expand culture,must remove from fermentation orpotential for flavor problems, musthandle bags several times a day.


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Genitically Modified Yeast

Saccharomyces cerevisiae with bacterial

malolactic gene implantedDeveloped by Hennie JJ Vuuren atU. of British Columbia, 2001Converts malic acid to lactic acid +CO2Very new product, not muchinformation availableMust use low inoculum the gene is

inactivated by ethanol


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Genetically Modified ML01 Yeast

Pros & ConsPros:

Easy, lactic acid complexity, toleratesSO2 & very low pH,Cons:

Timing of malic metabolism is critical,may still have to use bacterialinoculation if malic is not depleted.


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Freeze Dried Starter Culture Survival

During StorageSurvival during storage at different temperatures.

0

20

40

60

80

100

120

0 5 10 15 20 25 30Time (months)

S u r v

i v a l

( % )

4C

minus 18C30C

guaranteed stability


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Lab Evaluation of MLFPaper Chromatography:

EasyInexpensive ($40-45$/kit)Moderately accurate

Enzymatic Analyses:

DifficultExpensive ($25/test)Very Accurate

Reflectoquant type systemsSomewhat difficultExpensive ($500 +$1/test)

Varying Accuracy


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Paper Chromatography

Commercial Malolactic Cultures


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X10 differentcultures

Lallemand, Inc.Dist by: Scott Labs(707) 765-6666www.scottlaboratories.com/home.asp

XX5 different strainsVinquirywww.vinquiry.comTel: 707-838-6312

XXX

Viniflora CH-35Viniflora CH-16Viniflora Oenos

Chrs. Hansen, IncDist by: Gusmer EnterprisesPhone: (715) 258-5525www.filtermaterials.com/Gusmer/index.asp

FreezeDriedLiquidCultureProducer



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XLeuconostoc

oenos

BioSource Flavors, Inc.Muskego, WI 53150-0777Phone: 414 422 9085www.biosourceflavors.com/contact.htm

XX

44-4054-40

Pickering Winery Supply(415) 474-1588

http://www.winerystuff.com/

FreezeDried

LiquidCultureProducer



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XML-01 YeastStrain(Geneticallymodified)

American Tartaric ProductsLarchmont, NY 10538Phone: 914-834-1881

http://www.americantartaric.com/

XProMalicYeast Strain

Vinquiry(Lallemand product)

Tel: 707-838-6312www.vinquiry.com

FreezeDried

LiquidCultureProducer


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2005 Frontenac Wine Samples

WINE:

RS: 0.3%pH: 3.9TA: 0.735%VA: 0.07%

ETOH: 12.8%MLF (chromatography) Complete

HARVEST :

Brix 24: pH 3.4; TA 1.05

Sample# 1 Frontenac+ Oak & MLF

WINE:

RS: 0.2%pH: 3.74TA: 0.953 %VA: 0.04%

ETOH: 13.7%MLF (chromatography) Negative

HARVEST :Brix 24: pH 3.4; TA 1.05

Sample#2 FrontenacStandard Red Method


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ma lo lactic fermentation

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