REDUCING SULPHITES CONTENT IN WINES

Consumers and sulphites in wine

Roles and impacts of SO 2 in Oenology Bacteria Yeast Oxygene, quinones Tyrosinase, laccase Antiseptic Antioxidant Antioxidasic Oxidised SO 2 = sulfate (SO 4 2- ) Oxygen quinones Molecular SO 2 (H 2 SO 3 ) High ethanol Low ph High T Salified SO 2 (HSO 3- ) Aldehydes ketones sugars Bound SO 2 Free SO 2 total SO 2 Hardness Dryness Organoleptic impacts SO 2 odour Covering of fruity aromas Blocking of aldehydes (limitation of oxidised character) Yeast : production of SO 2 /acetaldehyde/h 2 S Metabolic effects temperature, turbidity, nutrition Human : toxicity, allergenicity

«Diversity is the place of art» Albert Camus Microbiological cartography of grape must DIVERSITY OF FLORAS: RISKS AND BENEFITS

Which flora on grapes? Vintage 2013 Hand harvest (nearly 20 kgs) Healthy grapes from organic parcels (Pinot noir x2, Chardonnay and Sauvignon) Direct pressing Divided in 2 deposits of 15 L Addition of sulphite 5 g/hl or none Every operation carried out with sterile equipment.

Which flora on grapes? Photographs of surface after 9 days at 20 C. Estate 1 Estate 2 Parcel 1 Parcel 2 Parcel 3 Parcel 4 Molds is growing quickly in surface. AF hasn t triggered.

Which flora on grapes? Alcoholic fermentation 50% 45% 40% 35% 30% 25% 20% 15% 10% 5% 0% State of AF after 9 days at 20 C. 60% : AF< 5% 40% : AF between 5 and 30% 0 10 20 30 40 50 60 70 80 80 samples of grapes

Which flora on grapes? Alcoholic fermentation 100% 90% 80% 70% 60% 50% 40% 30% 20% 10% 0% State of AF after 18 days at 20 C. 50% : AF< 20% 42.5% : AF between 20 & 50% 7.5% : AF> 50% 0 10 20 30 40 50 60 70 80 80 samples of grapes

Which flora on grapes? Micro-organisms on (healthy) grapes Grapes are contaminated by mold. Grapes are contaminated by yeasts : - Presence of yeast with low fermentative power and high potential of acetic acid production (such as Hanseniaspora). - Presence of yeast with very low fermentative power and with very low potential of acetic acide production (such as Metschnikowia). - Very low presence of yeast with strong fermentative power (such as Saccharomyces).

Diversity of micro-organisms on grape Molds and yeast Metagenomic survey on merlot grapes (according to Bauquis, 2017)

Yeast diversity in must : a double component Often majority (>70%) Kloeckera Hanseniaspora? Candida Pichia S. cerevisiae Metchnikowia Grape flora at maturity Flora present on winery equipment: Saccharomyces Candida Brettanomyces Variable levels of population: 10 3 to 10 6 cell/ ml

Evolution of yeasts floras Vine Must, beginning of AF Wine during AF >6% alcohol End of AF, wines Resistant to SO 2 and alcohol According to Blondin, 18 mars 2011, matinée technique des œnologues (Montpellier SupAgro )

Acetic acid producing yeast The specific case of cold soak

The specific case of cold soak Growth of Hanseniasporauvarum in must. Must of pasteurized Pinot noir : sugars 230 g/l, ph 3.20, no SO2 Incubation at 15 C Yeast (cell./ml) T0 T 1 day T 6 days Control (non contaminated) < 10 < 10 < 10 Hanseniaspora uvarum (contaminated) 320 22 000 70 000 000

The specific case of cold soak Activity of Hanseniasporauvarum in must. Must of pasteurized Pinot noir : sugars 230 g/l, ph 3.20, no SO2 Cold soak at 15 C Yeast addition (Sac.c.) at T7 days AF at 20 / 24 C. Acetic acid (g/l) End cold soak (T 7 days) End AF (T 14 days) Control 0.02 0.35 Hanseniaspora uvarum* 0.31 0.67 * Hanseniaspora produces nearly 10 times more ethylacetate thansaccharomyces.

Impact of temperature Low temperatures can promote non-saccharomyces (but also Saccharomyces uvarum). At low temperature (15 C), apiculated yeast resist better to alcohol. There are cases of dominance of apiculated yeast at the end of AF! To reason together with the level of SO 2. On the opposite, high temperatures (28 C) promote S. cerevisiae. According to Blondin, 18 mars 2011, matinée technique des œnologues (Montpellier SupAgro )

Yeast diversity in fermentation: benefits Potential production of metabolites of interest: Specific fruity esters Varietal thiols Aromatic complexity Other aromas Glycerol Specific fermentative capacities of some non Saccharomyces strains (osmotolerance, cryophily )

Yeast diversity in fermentation: dangers Potentially high production of acetic acid and ethylacetate. Potentially high production of H2S (linked to the nutrition).

Potential of acetate production Formation of volatiles during AF Volatile acidity Ethylacetate Alcohol reached: According to Blondin, 18 mars 2011, matinée technique des œnologues (Montpellier SupAgro )

Yeast diversity in fermentation: dangers Potentially high production of acetic acid and ethylacetate Potentially high production of SO 2 and/or acetaldehyde

Potential of production of SO 2 by yeast mg/l 45 40 35 30 25 20 15 10 5 0 Total SO 2 after AF RA17 S1 S7 S24 S25 S34

Variabilityof production of acetaldehyde A B C D E F G H Yeast strain According to Cheraiti et al, 2010

Yeast diversity in fermentation: dangers Potentially high production of acetic acid and ethylacetate Potentially high production of SO 2 and/or acetaldehyde Possible production of volatile phenols (Brettanomyces bruxellensis, Pichia guillermondi)

Consommation de la thiamine par K. apiculata [ 14 C-Thiamin] (µg l -1 ) 6 0 5 0 4 0 3 0 2 0 1 0 0 0 1 2 3 T i m e ( h )

And bacteria? Sometimes, stronger contaminations

And bacteria? 0,8 Production of acetic acid after contamination of the must with acetic bacteria Pasteurized must of pinot noir - inoculation at T0 (104 cell/ml) - prefermentative cold soak at 10 C or 16 C during 7 days then inoculation in S. cerevisiae yeast Acetic acid (g/l) 0,7 0,6 0,5 0,4 0,3 0,2 0,1 T0 T 7 days T 14 days T 21 days 0 10 C 16 C 10 C 16 C Acetobacter Gluconobacter

Fermentation withlocal flora: balance benefits/risks

Microbiological risks and sulphiting Skin maceration or cold soak Pressing Settling of the must Racking off Grapes : grape-gondola or harvesting machine Prefermentative microbiological risks: Kloeckera apiculata (Hanseniaspora uvarum) Brettanomyces bruxellensis Some bacterial risks AF triggering by indigeneous S. cerevisiae Alcoholic fermentation Beginning AF 1/3 AF MLF End AF Ageing and storage Bottling

Metschnikowia fructicola Gaïa TM PRE-FERMENTATIVE BIOCONTROL

A first approach: split addition of yeast 1,00E+06 Non Saccharomyces yeast populations potentially contaminating (ufc/ml) - pinot noir - potential alcohol: 12,9% vol - ph wine=3,44 - countings before the inoculation carried out after cold soak 1,00E+05 Population (ufc/ml) 1,00E+04 1,00E+03 1,00E+02 1,00E+01 1,00E+00 Early inoculation (20 g/hl at the filling of the tank) Split inoculation (5 g/hl at the filling then 15 g/hl after cold soak) non Saccharomyces yeasts Late inoculation (20 g/hl after cold soak)

M. fructicolaand biocontrol : an old story? Controls M. fructicola Wound then inoculated apples : - 1 st series with sterile water (10µL) - 2 nd series with a suspension of M. fructicola (5.10 7 cells/ml) 2 hours after: inoculation of both series with Penicilium expansum then kept at 25 C during 4 days. Liu et al, 2011 in FEMS Microbiology Ecology

MetschnikowiafructicolaGaïa TM No fermentative power Low nutritional needs Antimicrobial activity, especially anti-kloeckera Excellent implantation and survival Positive sensory contribution No production of undesirable metabolites

M. fructicola(gaïa TM ): A true prefermentative tool Merlot at 14 C from T=0 to 96h, then increase to 24 C Metschnikowia : 25g/hL at T=0, then S. cerevisiae in sequential inoculation at T=96H Control S. cerevisiae : 25g/hL at T=0 120 100 80 T=0 inoculation Control: S. cerevisiae Gaïa: Metschnikowia T=96H Modality Metschnikowia: S. cerevisiae inoculation CO2 (g/l) 60 40 Gaïa S. cerevisiae 20 0 0 50 100 150 200 250 300 350 Time (hours) Raynal et al, 2014

Implantation and survival of M. fructicolagaïa TM On must at low temperature and on long-term 1,0E+08 Populations of Metschnikowia (cfu/ml) depending on the inoculation temperature from 0 to 80 days (muscat stored at 0 C) 1,0E+07 1,0E+06 1,0E+05 1,0E+04 1,0E+03 0 C 5 C 10 C 1,0E+02 1,0E+01 1,0E+00 0,0 0,1 12,0 30,0 40,0 50,0 60,0 80,0 Time (days)

M. fructicola(gaïa TM ): biocontrol against Kloeckeraand volatile acidity 0,80 Production of acetic acid by Kloeckeraapiculata wih or without Gaïa TM in a must (sugars 230 g/l, ph3.20, no SO2, pasteurization) - (SD: 0,05 g/l) values given after AF 0,70 0,60 Acetic acid (g/l) 0,50 0,40 0,30 0,20 0,10 0,00 Non contaminated control Contamination with Kloeckera* Contamination with Kloeckera + biocontrol with Gaïa Gerbaux et al, 2015

M. fructicola(gaïa TM ): biocontrol and sensory contribution in cold soak Sensory evaluation at the end of ageing of a red wine (pinot noir) in tanks of 2,5 hl with cold soak, with o withoutmetschnikowia - Average values on 2 years. Fruits Intensity 6,0 5,5 5,0 4,5 4,0 Gaïa Metschnikowia B Control Global quality Acidity / Roundness Balance

Gaïa: a tool of biocontrol against Botrytis cinerea B. cinerea: contaminating agent on desiccated grapes Gaïa fights actively against it : Production of pulcherriminic acid S. cerevisiae

MetschnikowiafructicolaGaïa TM Preservation of desiccated grapes (raisining) Competitive asset Goal: to limit the post-harvest growth of Botrytis cinerea, for desiccated grapes.

MetschnikowiafructicolaGaïa TM Preservation of desiccated grapes (raisining) Competitive asset Gaïa 50 g/ ql: 41 days of desiccation

Management of SO 2 and SO 2 -binding compounds IOC BE YEAST: FERMENTATIVE BIOCONTROL

IOC BE yeast : interest Guaranteeing a tool for controlling SO 2 levels in wines: By zero production of SO 2, and independently of the conditions By a very low production of acetaldehyde, which combines SO 2 Consequences: clean wines

How does yeastworkwith sulfates and sulphites? Homoserine SO 4 2- SO 2 Homoserine SO 4 2- SO 2 Identification of a low (not) SO 2 / acetaldehyde / - producing strain Homocysteine High sulphite-producing strain Homocysteine Low sulphite-producing strain Increased metabolism if: - High NH 4 + - Low temperature - Presence of sulfates - Addition of sulphites in must

Birthof a toolto decrease sulphites in wine Breeding Enhancement Yeast of enological interest «Low SO 2» mother-yeast Successive back-crossings Results (after a lot of oenological validations): a new yeast, containing a big part of the heritage from IOC yeast of enological interest, but with the guarantee of no production of SO 2 whatever the external conditions are.

Zero production of SO 2 whatever the conditions are Differences between added SO2 and total SO2 analysed in finished wine (ppm) 60 50 40 30 20 10 0-10 -20 Production of SO 2 : differences between added SO 2 and total SO 2 found in wine Reference yeast A * * Other reference yeasts (* = additional yeast not included in the trial) grenache rosé (added SO2 30 mg/l - ph 3.30 - alcohol 14 % vol) sauvignon blanc (added SO2 50 mg/l - ph 3.27 - alcohol 12.5 % vol) sauvignon blanc (added SO2 40 mg/l) sauvignon blanc (added SO2 65 mg/l -ph 3.42 - alcohol 12.8 % vol) sauvignon blanc (added SO2 35 mg/l - ph 3.46 - alcohol 12.6 % vol) colombard (added SO2 50 mg/l - ph 3.42 - alcohol 12.8 % vol) IOC BE THIOLS

Low production of acetaldehyde less bound SO2 IOC BE FRUITS: decrasing the acetaldehyde content (deviation between concentrations obtained with IOC BE FRUITS and those ones obtained with reference yeast 0% -10% -20% -30% -40% -50% -60% -70% Chardonnay Maccabeu Grenache/cinsault

LEES AND PRESERVATION OF WINE QUALITIES

Mechanisms of oxidation GSH (reduced glutathione) Trapping of sulfur compounds (including fruity thiols) Formation of aldehydes through Strekker degradation Polymerizing reactions (browning)

Anticipating the protection against oxidation: the impact of inactivated yeast rich in glutathione Principle : Optimizing richness in antioxidants in musts and specially in wines Formulation : Specific inactivated yeast, naturally riche in reduced glutathione Goals : Increasing biodisponibility of reduced glutathione in wines (and must) in order to induce the resistance of aromas to oxidation.

Pay attention to the different chemical species of glutathione! Optimization of production process in order to increase the synthesis of GSH by yeast before inactivation Optimization of the content in reduced (or true) glutathione compared to total glutathione (GSH+GSSG (oxidized glutathione)). Kritzinger et al., 2012 Amounts in reduced, oxidized and total glutathione of different inactivated yeast naturally rich in glutathione. Produit 5 Despite an apparently higher concentration in total glutathione, product N 3 is however less rich in reduced glutathione, the only one efficient to protect wine against oxidation.

Alternatives to lees to protect wines against oxidation 3,00 Impact of Glutarom Extra added at the beginning of AF on the content of reduced glutathion in a wine with low SO2 additions (4-15 mg / L) 2,50 Concentracion (mg/l) 2,00 1,50 1,00 0,50 0,00 sauvignon 2014 chardonnay 2014 GLUTAROM EXTRA Control

A better resistance to air exposure Low SO2 conditions: evolution of yellow colour during air exposure - chardonnay 2014 analysis after AF after SO2 addition SO2 additions: on must: 0 g/hl wine post AF + before bottling : 0,4 g/hl 0,700 0,600 0,500 DO 420 nm 0,400 0,300 0,200 0,100 0,000 0 10 20 30 40 50 60 70 80 90 100 Time of air exposure (hours) GLUTAROM EXTRA Control

Anticipating the richness in reduced GSH In low SO2 wines, GLUTAROM EXTRA permits amounts in GSH similar or higher than the ones obtained with a full dosage of SO 2 addition (added at the settling of the must then post AF). Glutarom These results are obtained with an addition at the beginning of AF.

Thanks for your attention!