Good fermentations gone bad. Why? Sigrid Gertsen-Briand Ohio, February 12 th, 2008

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1 Good fermentations gone bad. Why? Sigrid Gertsen-Briand Ohio, February 12 th, 2008

2 Without yeast, it s just juice! Two healthy cells are better than one ill-prepared cell!

3 Factors affecting Fermentation Management - Key Interrelationships JUICE or MUST TEMPERATURE YEAST STRAIN CELL NUMBERS & HEALTH MAXIMUM FERMENTATION MANAGEMENT NUTRITIONAL FACTORS TOXIC FACTORS COMPETITIVE FACTORS

4 What do winemakers want? SECURE FERMENTS sugars regular fermentation = easy finish absence of metabolic off-flavors Good fermentation: slow or fast, but good finish Acceptable fermentation: slow but right to the end Worst case: fast at the beginning and sluggish / stuck at the end time Key parameter: slope at the end

5 Good Alcoholic Fermentation Practices (GFP)

6 If Good Fermentation Practices are used... Sugar if not...higher risk of sluggish or stuck alcohol fermentation finish Fermentation length

7 Rehydration!!!!

8 PROPER YEAST REHYDRATION Clean water at 110 o F (or 104 o F if not using GO-FERM) & HANDLING 1 st suspend GO-FERM 2 nd add Active Dried Yeast Suspend GO-FERM 110 o F Add Active Dried Yeast minutes JUICE Avoid cold shock >18 o F Add yeast slurry to bottom of tank

9 Some factors to consider when catering to the little critters Rehydration Cell population Nutrients Oxygen ph Particulate matter/gravity Pressure / CO2 Sugar / osmotic pressure Alcohol toxicity Wild yeast, bacteria & toxic substances Temperature Fructose

10 Good Fermentation Practice (GFP) at yeast rehydration stage: Addition of micronutrients Directly feed selected yeast with balance of key vitamins and minerals Why? They won t be used by ambient microbes, or bound by must components Helps yeast viability and vitality

11 Yeast Nutrient Development

12 0,9 Effect on Fermentation Kinetics of GO-FERM Micronutrient Addition During Yeast Rehydration A. Julien, J. Sablayrolles - INRA Montpellier 2001 dco2/dt (g/l.h) 0,8 0,7 0,6 0,5 0,4 0,3 0,2 0,1 0 30g/hl GO-FERM added at rehydration Uvaferm CEG inoculated at 25g/hl into MS 70 medium CO 2 evolution at 24 o C Greater degree of slope indicates Stronger fermentation finish 43 slope Control sluggish fermentation 19 slope Time (h)

13 MICRONUTRIENTS: Minerals Magnesium better alcohol, temperature and osmotic resistance, ratio Ca:Mg < 1, Zinc cofactor of glycolysis enzymes, increase alcohol tolerance regulation of by-products (esters, alcohols, fatty acids), Manganese synergistic effect with Zn, shorter generation time Copper essential element, but toxic above 1-2 mg/l Potassium must be > 300 mg/l at low ph s

14 Why is Mg so Important? % viable viabilité après cells 60 after min stress 60 (%) minutes Yeast Alcohol Tolerance! 0% 5% 10% 15% 20% 2 mm Mg 50 mm Mg Viability of S. cerevisiae after 60 min of Ethanol level at different concentrations of Mg 2+ (Birch and Walker, 2000)

15 MICRONUTRIENTS: VITAMINS Pantothenate avoids H 2 S and VA formation, better kinetics, less acetaldehyde, strain sensitivity Biotin better kinetics, synergic effect with N, increases ester production, higher yeast viability at end AF Thiamine Inositol better cell growth, less acetaldehyde and VA essential for membrane phospholipid synthesis

16 Why is Pantothenic Acid Important? 6 60 mg/l YAN 250 mg/l YAN total H2S produced during fermentation (mg/l) Avoid H 2 S! Pantothenic acid (μ g/l) Production of hydrogen sulphide by S. cerevisiae in a synthetic juice at different concentrations of Yeast Assimilable Nitrogen and Panthotenate (WSU, C. Edwards 2001)

17 Yeast Nutrient Validation

18 Yeast Viability Viability: 70% = 49 x 10 6 live cells Viability: 43% = 23 x 10 6 live cells Control 18 C Goferm 18 C Hours Similar results at 24 C and 28 C, there is a strong positive effect on yeast viability when Go-Ferm (minerals and Vitamins) added during yeast rehydration.

19 Nutrient Trial in Napa Valley Chardonnay Effect of nutrients on YAN utilization Control DAP Nutrient 1 + DAP Nutrient 2 Nutrient 3 Go-Ferm PDM standard FAN (mg/l) Time (days)

20 Good Fermentation Practice (GFP) at yeast rehydration stage: Yeast Inoculation Level In high potential alcohol musts add higher yeast inoculums to avoid dilution effect.

21 Yeast viability during alcoholic fermentation (cfu/ml) > million CFU/mL 2-4 million CFU/mL 2-4 million CFU/mL Fermentation time

22 Good Fermentation Practices should be used during > million CFU/mL 2-4 million CFU/mL 2-4 million CFU/mL Fermentation time

23 > million CFU/mL Higher yeast inoculation rate lowers dilution of the initial yeast cells survival factors 4-8 million CFU/mL 2-4 million CFU/mL 2-4 million CFU/mL Survival factors are important to ensuring the proper working of the cellular membrane: poly-unsaturated fatty acids and sterols

24 Good Fermentation Practice (GFP) at yeast rehydration stage to protect yeast under high brix and more difficult fermentation conditions : Addition of unsaturated fatty acids & sterols Protect yeast against initial osmotic shock lower V.A. Build-up yeast cell wall content of yeast stress resistant factors protect against ethanol toxicity

25 Yeast Cell Yeast Cell Wall Cross section

26 Yeast cell wall composition: Plasma Membrane is ~5% lipids (sterols & unsaturated fatty acids)

27 After yeast inoculation and lag phase begins yeast exponential growth phase 2-4 million cfu/ml Inoculation rate 2 lbs. per 1000 gallons (25g/hL)

28 Yeast exponential growth phase 4-8 million cfu/ml Plasma Membrane now is ~2.5% lipids (sterols & unsaturated fatty acids)

29 Yeast exponential growth phase 8-16 million cfu/ml Plasma Membrane now is ~1.25% lipids (sterols & unsaturated fatty acids)

30 Yeast exponential growth phase million cfu/ml Plasma Membrane now is ~0.6% lipids (sterols & unsaturated fatty acids)

31 Yeast exponential growth phase million cfu/ml Plasma Membrane now is ~0.3% lipids (sterols & unsaturated fatty acids)

32 Yeast exponential growth phase million cfu/ml Plasma Membrane now is ~0.15% lipids (sterols & unsaturated fatty acids)

33 Yeast cell wall composition: Plasma Membrane is now <0.2% lipids (sterols & unsaturated fatty acids) A critically low level!

34 To help avoid lipid depletion, add them during yeast rehydration Rehydration Without Protection ADY membrane «White colloid» Rehydration with UFA & Sterol NATSTEP Protection

35 H + H + ATPase Sterols and fatty acids H + Structural proteins ATP ADP ATP ADP Transport protein H + Enzymatic proteins [H + ] [H + ] ph int. = 5-6 Alcoholic fermentation ph ext. = 3-4

36 Yeast Preparation for high Brix musts During Rehydration Suspend 1.25 parts NATSTEP stimulant and protectant Suspend 1 part active dried yeast 110 o F

37 DAP AMMONIA NUTRITION It s nice to have a dessert to complete the meal. but Could you just live on sweets?

38 DAP : what a wonderful world!!!? cheap source of easily assimilable nitrogen BUT must be used in combination with oxygen must be added at 1/3 sugar depletion otherwise big biomass and even worse starvation at the end cannot be used in massive doses harsh mouth at too high levels and legal limits risk of VA increase completely adsorbed by the yeast in few hours more H 2 S formation when no nitrogen available

39 DAP is used very quickly Rate of H 2 S liberation (mmol/g dry wt/h) NH NH Jiranek, 2000 hours In nitrogen deficient musts the addition of DAP gives more biomass, which is then starving and producing even more H 2 S 21

40 Don t just feed DAP yeasts benefit from a balanced diet too!

41 YANC OR YAN Yeast Available Nitrogen Content sum of assimilable nitrogen from Free Ammonia Nitrogen (FAN) plus the assimilable nitrogen from alpha amino acids low levels associated with production of undesirable sulfide compounds and stuck fermentations

42 INTEGRATED NUTRITIONAL STRATEGY FOR WINE YEAST JUICE YANC rehydration end of lag 1/3 AF HIGH N > 225 mgn/l Go-Ferm lb/kgal MEDIUM N > 125 mgn/l Go-Ferm FERMAID K < 225 mgn/l 2.5lb/kgal 2lb./kgal LOW N < 125 mgn/l Go-Ferm DAP FERMAID K 2.5lb/kgal 2.5lb/kgal 2lb/kgal or more

43 Go-Ferm & FERMAID FERMAID K: ARE THEY USELESS NOW? In high sugar - nitrogen deficient musts a YAN addition (at 1/3 AF) is still needed Go-Ferm FERMAID K DAP provides ab. 10 mgn/l at 30 g/hl (100% a-amino) provides ab. 30 mgn/l at 30 g/hl (mix of a-amino and ammonia) provides ab. 60 mgn/l at 30 g/hl (100% ammonia)

44 Effect of Fermaid to decrease sulfur off-flavours Témoin PDA (2 fois) Fermaid à Intensité analyse sensorielle Odeurs soufrées Ananas Acétate d'isoamyle Volume Rugosité

45 GFP at beginning of fermentation: red musts Addition of oxygen as soon as the cap is formed (usually 15 g/l sugar fermented)

46 5 to 10 mg/l dissolved oxygen O CO 2 2

47

48 Move your yeasts! Delestage Complete draining of fermenter O 2

49 From a yeast point of view Nutrient pool limited Low ph: more difficult for yeast for nutrient uptake, biomass production and when combined with other factors (low temperature).

50 H + H + ATPase Sterols and fatty acids H + Structural proteins ATP ADP ATP ADP Transport protein H + Enzymatic proteins [H + ] [H + ] ph int. = 5-6 Alcoholic fermentation ph ext. = 3-4

51 Where is the H 2 S coming from? SO 2-4 SH 2 cisteína ATP ATP SO - 4 APS PAPS HSO 3 3 NADPH ADP ADP 3 NADP SH 2 O-AS O-AH Nitrógeno intra celular HSO 3 - metionina

52 GFP during peak of fermentation: red musts Maximum temperature reached under the cap Cap during the fermentation Fermenting Juice Yeast Biomass

53 Don t grow yeast up too fast temperature management Low alcohol concentration High alcohol concentration Slow alcohol excretion

54 Reds, under the cap, peak temperature maximums relative to the initial osmotic shock (in warm or hot climate regions) 20 Brix 21 Brix 22 Brix 23 Brix 24 Brix or more 95 F 90 F 85 F 80 F 76 F It integrates warm or hot climate grapes constraints for the yeast

55 GFP during the last quarter of fermentation: red musts Regular movement of the yeast during their death phase Deep punching down Bring the yeasts back up into suspension

56 Without yeast, it s just juice! Two healthy cells are better than one ill-prepared cell!

57 Stuck Fermentation Restart Protocol

58 What to do in case of stuck fermentation 1. protect and prepare the stuck wine 2. prepare the yeast 3. Re-start the fermentation

59 1. Protection-preparation of stuck wine Avoid Oxidation Development of spoilage Micro-organisms (acetic acid and lactic acid bacteria)

60 What to do 1 Analyse the wine: ph, alcohol, residual sugars, VA, free and total SO 2 2 Rack the wine avoiding air contact, to eliminate the lees May May contain contain substances substances responsible responsible for for spoilage spoilage LEES Carriers Carriers of of undesired undesired micro-organisms micro-organisms May May contain substances which which are are toxic toxic for for the the yeasts yeasts

61 What to do 3 Add SO 2 according to the analysis results 4 Top off the containers carefully 5 Keep the wine temperature at around 20 C 6 Filter (if possible) to avoid spoilage

62 What to do 7 Add inactive yeast residues (yeast hulls) to adsorb toxic substances for yeasts (C 8, C 10 and C 12 fatty acids) Yeast hulls g/hl g/hl Keep in contact for hours, stirring lightly once in a while Let the yeast residues settle out rack or filter Add FERMAID K g/hl g/hl In the most difficult cases + Cellulose g/hl g/hl

63 2. Yeast preparation Protocol Based on 100 hl of stuck wine or must With: 12 % alcohol 15 g/l of residual sugars

64 PROPER YEAST REHYDRATION FOR RESTARTING 100hL STUCK WINE 50 L Clean water 110 o F Suspend 5 kg GO-FERM Wait until suspension temperature drops to 104 o F before adding 5kg rescue yeast such as Uvaferm 43 1 st suspend GO-FERM 2 nd add Yeast Light mixing to break up any clumps minutes DO NOT WAIT LONGER! Go to the next step

65 Yeast preparation Adjustment to the alcohol content Add the 120 L to: Add the 60 L to: rehydrated yeast 20 L stuck wine 30 L water 10 Kg sugar 25 g of FERMAID K keep at 25 C for about 6-8 hours Mix once in a while 0 % alcohol 2,4 % alcohol g/l sugars DO NOT WAIT MORE THAN 8 hrs!

66 add the 120 L to: Yeast preparation Adjustment to the alcohol content Add the 500 L to: Add the 10 hl to: 200 L stuck wine 100 L water 20 Kg sugar 250 g of FERMAID K keep at C for about hrs Check for the occurrence of fermentation 500 L of stuck wine Keep at 20 C for about hrs Check for the occurrence of fermentation 5 % ALCOHOL ATTENTION! Sometimes longer times are needed 8,5 % ALCOHOL g/l sugars g/l sugars

67 3. Add the 10 hl to: Fermentation re-start INOCULATION TEMPERATURE avoid temperature below 65 F (18 C) if necessary, warm up to C 90 hl of stuck wine TIME from 5 to 20 days sometimes longer than 20 days Fermentation re- start until the residual sugars exhaustion

68 Warning! Very important parameters to succeed in restarting a stuck fermentation Yeast quantity used for the inoculation (at least 10 million cells/ml - 50 g/hl of wine) Physiological yeast conditions: adjustment to alcohol is indispensable Analytical wine characteristics (evaluate the risks and the difficulties of re-starting) Yeast strain choice for the inoculation: - It s better to avoid the same yeast strain used at the beginning - It is very important the rapidity of fermentation re-start Keep the the cellar very very clean, wines with with residual sugars are are more sensitive to to microbial spoilage

69 Pro-Restart!! ALCOHOL- ACCLIMATIZED IMMOBILIZED YEASTS TO SOLVE STUCK OR SLUGGISH FERMENTATION PROBLEMS

70 Why Pro-Restart? Works quickly due to its preconditioning and direct addition into a stuck or sluggish fermentation. Handles easily compared to traditional restart methods. Decreases spoilage risks related to microbiological contamination (acetic or lactic bacteria, Candida mycodermi and Brettanomyces) due to its rapid fermentative activity and helps avoid excessive SO2 additions.

71 Why Pro-Restart? Decreases risks related to color oxidation and flavor loss caused by sluggish fermentations. Avoids blending a stuck wine with other fermenting wines. Preserves the economic value of the wine due to rapid sugar consumption without volatile acidity increase or other spoilage problems. Respects local regulations on maximum authorized residual sugars. Avoids addition of excess nutrients for potential spoilage problems.

72 THANK YOU! For more information or