Peter Salamone, PhD Technical Manager – North America

1. Peter Salamone, PhD Technical Manager – North America

2. General Trends Vintage 2012 • Ideal Growing Season • Record Harvest – High Quality Fruit • Low YAN Compared to Previous Harvests • More AF Issues than MLF

3. 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

4. Yeast Nutrition Yeast Rehydration Survival Factors Assimilable Nitrogen Vitamins Minerals The most important and indispensable factors for a good fermentation and aromatic quality of the wines

5. 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

6. 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 8Major Minerals - Ca, Co, Cu, Fe, Mn, Mo, Ni, Zn Mg K Macro nutrients >100 ppm All factors are important but… Supplement additions are usually based on YAN alone So… Balancing Nitogen does not always balance total nutrition ! ! Micro nutrients <1 ppm Trace Nutrients – Se, B, Na, Inositol, ???

7. Unhappy Yeast Consequences • Stuck or Sluggish Fermentations • Elevated C8 + C10 Fatty Acids • High SO2 • H2S – Other Aroma Consequences

8. Factors Influencing MLF • Ethanol • pH • FSO2 • TSO2 • Temperature • Malate or < ~1 g/L • Polyphenols undetermined • 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

9. C8+C10 FA World Survey Average levels of fatty acids measured post- AFaccording to the country of origin. 282 wines, 3 vintages, red and white, North and South Hemisphere.

10. C8+C10 FA vs MLF 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).

11. C8+C10 FA vs MLF 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.

12. 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

13. C8+C10 FA vs MLF • ? 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

14. YAN Effect on FA

15. 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

16. Folpet Effect on C8+C10 FA Folpet is a protective leaf fungicide Indirect effect on MLF through elevated FA >25 mg/L C8+C10 FA can inhibit MLF

17. Boscalidvs MLF 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

18. Dimethomorphvs MLF 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.

19. 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

20. Co-Inocculationvs Sequential Choosing the type of inoculation to implement:

21. Co-Inocculation – For You? Alcoholic fermentation Malolactic Fermentation Pre-fermentative maceration Sequential inoculation Late 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 Early co-inoculation

22. Thank You! Peter Salamone, PhD Technical Manager, NA Laffort USA 1460 Cader Lane, Suite C Petaluma, CA 94054 (707 934-5771 Peter.Salamone@Laffort.com