Peter Salamone, PhDTechnical Manager – North America

General Trends Vintage 2012

• Ideal Growing Season

• Record Harvest – High Quality Fruit

• Low YAN Compared to Previous Harvests

• More AF Issues than MLF

YAN and Fermentation

• YAN values dropped from ~205 to ~145

• Nitrogen/Nutrient Supplementation– DAP alone– Complex Nutrients– Organic Nutrients– Yeast Rehydration Survival Factors

• No Pattern to Stuck and Sluggish Ferments

Yeast Nutrition

AssimilableNitrogen

Vitamins

Minerals

The most important and indispensable factors for a good fermentation and aromatic quality of the wines

Yeast Rehydration

Survival Factors

Where to Look ?

• Poor Fermentation Causes – No Shortage!– Low Population of Viable Yeast– Fermentation Temperature Spikes – Heat, Cold– Microbial Competition – Early, Late– Toxins – Microbial, Al, Pb, Pesticide Residues– Yeast Genetic Mechanisms – Prions - Epigenetics– Nutrition

• C / N Balance - Brix / YAN• Macro nutrient shortage• Micro nutrient shortage• Trace nutrient shortage

Yeast Nutrition

Yeast Growth Factors

Nitrogen YAN = Ammonia + alpha amino acids Vitamins

8 B complex vitamins - B1 – thiamine B2 – riboflavin B3 – niacin B5 – pantothenic acid B6 – pyridoxine B7 – biotin B9 – folic acid B12 - cobalamin Minerals

8 Major Minerals - Ca, Co, Cu, Fe, Mn, Mo, Ni, Zn

All factors are important but…

Supplement additions are usually based on YAN alone

So…

Balancing Nitogen does not always balance total nutrition

K Mg

Macro nutrients >100 ppm

Micro nutrients <1 ppm Trace Nutrients – Se, B, Na, Inositol, ???

! !

Unhappy Yeast Consequences

• Stuck or Sluggish Fermentations

• Elevated C8 + C10 Fatty Acids

• High SO2

• H2S – Other Aroma Consequences

Factors Influencing MLF

• Ethanol• pH• FSO2• TSO2• Temperature• Malate or < ~1 g/L• Polyphenolsundetermined• Nutrient Status may be proportional to initial YAN• AF stuck or sluggish• Microbial Status < 103 – 104 cells/mL• Fatty Acids C8 + C10 concentration

Many of the values depend upon the

specific Oenococcus strain being used

C8+C10 FA World Survey

Countries C8 (mg/L) C10 (mg/L)

England 61.5±27.4 14.5±6.0

Portugal 34.8±18.6 8.7±5.6

Switzerland 24.8±18.7 7.8±1.8

Italy 24.4±14.2 7.5±3.5

Germany 23.9±8.5 6.4±2.7

France 21.9±12.9 4.5±1.0

China 18.0±8.7 1.1±0.2

USA 12.2±7.0 2.8±1.4

Spain 11.4±2.3 2.5±0.5

Chile 8.1±5.8 1.9±0.8

South Africa 3.9±1.2 1.9±0.5

Average levels of fatty acids measured post- AF according to the country of origin. 282 wines, 3 vintages, red and white, North and South Hemisphere.

C8+C10 FA vs MLF

0

10

20

30

40

50

C8 C10

C8 a

nd C

10 co

ncen

tratio

ns (m

g/L)

Figure 2: Of the 282 wines studied: 156 presented no problems with MFL (group I in green) and showed average levels of C8 (9.0 ± 5.0) and C10 (2.1 ± 1.1) relatively low. The 126 wines in which MLF was problematic (group II in pink) showed significantly higher average levels of C8 (34.4 ± 7.4) and C10 (9.3 ± 2.5).

C8+C10 FA vs MLF

0

0,5

1

1,5

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2,5

0 10 20 30 40 50

Days after inoculation

L-m

alic

aci

d (g

/L)

MLF monitored in a wine with C8 =5 mg/L and C10=2 mg/L* (♦), in the same wine with C8 =20 mg/L and C10=4 mg/L (●) and in the same wine with C8 =50 mg/L and C10=20 mg/L (■). Trials were made in triplicate. Malolactic starter is inoculated at the initial population of 106 cell/mL at day 0.

C8+C10 FA California 2011

• 225 random fermentation samples from across California

• Less than 5% above 15 mg/L C8+C10 FA

• All others below levels of concern

• Two problem sluggish MLF tested ~22 mg/L C8+C10 FA

• The problem MLF above were ~30 FA including C6

• Medium Chain FA assay at ETS Labs

C8+C10 FA vs MLF

Country Wine without MLF issues Wines with MLF issues

Number of wines analyzed

s C8 (mg/L) C10 (mg/L) Number of

wines analyzed C8 (mg/L) C10 (mg/L) France 48 12.9±2.1 2.7±0.6 44 37.3±9.7 10.6±5.7 Italy 22 11.2±1.2 3.4±0.4 22 38.4±8.4 12.6±6.6 South Africa 17 3.5±1.6 1.7±0.5 15 11.1±5.4 2.8±0.3 Australia 16 2.9±1.5 1.0±0.4 12 12.3±3.2 3.6±2.2

? MLF issues are not solely due to C8 +C10 Fatty Acid levels

? Native MLF by endogenous strains have varying tolerance for C8 +C10 Fatty Acid levels

? Undiscovered factors may reduce tolerance of C8 +C10 Fatty Acid levels

YAN Effect on FA

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10

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40

50

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75 125 175 225 275

Available nitrogen (mg/L)

C8 (m

g/L)

at

the

end

of t

he A

F

Phytosanitary Treatments and Effect on MLF

• Bordeaux 2010 vintage was very dry at end of season

• Washout by rain was minimal

• Led to persistence of abnormally high residues in must– Folpet residue

• Two phytosanitary compounds were >100 ug/L– Boscalid– Dimethomorph

Folpet Effect on C8+C10 FA

C8+C10 = f(Folpet µg/L)

0

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0 20 40 60 80 100 120 140 160 Folpet (µg/L)

C8+C10 (mg/L)

Folpet is a protective leaf fungicide

Indirect effect on MLF through elevated FA

>25 mg/L C8+C10 FA can inhibit MLF

Boscalid vs MLF

Parameters Values Temperature 20°C pH 3,7 ASV (% vol.) 12,4 Total SO2 (mg/L) 22 L-malic acid 1,8 Fatty acids (octanoic acid + decanoic acid in mg/L) 8 Brettanomyces (cells/mL using quantitative PCR analysis) <1/10 mL Lactic bacteria (cells/mL using epifluorescence) 2.106 cell/mL Boscalid (µg/L) 262

Chemical Name: 3-pyridinecarboxamide, 2-chloro-N-(4’chloro[1,1’biphenyl]-2-yl) Common Name: Boscalid (BAS 510) Trade Names: Emerald, Endura, and Pristine Chemical Class: Carboxamide aka anilide

Dimethomorph vs MLF

Tank Dimethomorph (µ g/L)

Number of days before MLF completion (from bacteria inoculation carried out 24h afterwards

each time)

Octanoic and decanoic acids (mg/L) at the end of

AF I Not detected 12 7 II Not detected 14 14 III Not detected 11 12 IV 92 54 8 V Not detected 10 5 VI 191,5 42 6 VII Not detected 18 12

Dimethomorph is a systemic fungicide which protects plants from molds, as well as killing molds on plants and preventing their spread. It is a cinnamic acid derivative and a member of the morpholine chemical family.

Dealing with Difficult MLF

PREVENTIVE

Correct Nitrogen Deficiencies in Must/Juice

Rehydrate Yeast with a Nutrient Containing Survival Factors

PROACTIVE

Utilize Co-Inocculation Strategy – Early (24 hr after yeast) or Late (~5 brix left)

After Determination of Elevated Levels of C8+C10 FA – Test Stuck/Sluggish for FA - Use FA Resistant Oenococcus Strain for MLF

REMEDIAL

Detoxify Wine by Yeast Hull FA Adsorption – MLF Restart Protocol

Co-Inocculation vs Sequential

Type of inocculation

Step Objective

Early co-inocculation

24-48 h after AF start-up

Time gain and fault preventionOptimized fermentation management

Late co-inocculation

At 3-5 brix remaining

Monopolization of the ecosystem and fault prevention. Secures a traditional vinification (AF then MLF)

Sequential inocculation

After AF,at pressing

MLF after post AF macerationMLF in barrels

Spring inocculation and/or restart

Later…Remedial

Restarting stuck MLFSpring MLF

Choosing the type of inoculation to implement:

Co-Inocculation – For You?

Pre-fermentative maceration

Alcoholicfermentation

MalolacticFermentation

Sequential inoculation

Late co-inoculation

Early co-inoculation

• Lower Stress on Oenococcus at Inocculation

• No latency between AF and MLF – control indigenous microflora

• Enococcus, a facultative heterofermenter, does not produce Acetic Acid from 6 carbon sugars-Glucose/Fructose

• Biogenic Amines – Strain Specific – Genetic Screening

Thank You!

Peter Salamone, PhDTechnical Manager, NA

Laffort USA

1460 Cader Lane, Suite CPetaluma, CA 94054

(707 934-5771Peter.Salamone@Laffort.com