REDUCING SULPHITES CONTENT IN WINES · 2018-09-03 · Vintage 2013 Hand harvest (nearly 20 kgs) Healthy grapes from “organic” parcels (Pinot noir x2, Chardonnay and Sauvignon)

REDUCING

SULPHITES

CONTENT IN

WINES

Consumers and

sulphites in wine

Roles and impacts of SO2 in

Oenology

total SO2

Tyrosinase, laccase

Free SO2

Antiseptic

Bacteria Yeast

Molecular

SO2(H2SO3)

Salified

SO2(HSO3

-)

Bound

SO2

Antioxidant Antioxidasic

Oxygene, quinones

SO2 odour

Covering of

fruity aromas

Blocking of aldehydes

(limitation of oxidised

character)

Oxidised

SO2 = sulfate

(SO42-)

Hardness

Dryness

Metabolic effects

Human :

toxicity, allergenicityYeast :

production of

SO2/acetaldehyde/H2S

High ethanol

Low pH

High T°

Oxygen

quinones…

Aldehydes

ketones

sugars

Organoleptic impactstemperature,

turbidity, nutrition

DIVERSITY OF FLORAS:

RISKS AND BENEFITS

Microbiological cartography of grape must

« Diversity is the place of art »

Albert Camus

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.


0%

5%

10%

15%

20%

25%

30%

35%

40%

45%

50%

0 10 20 30 40 50 60 70 80

Alc

oh

olic

ferm

enta

tio

n

80 samples of grapes

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%

90%

100%

0 10 20 30 40 50 60 70 80

Alc

oh

olic

ferm

enta

tio

n

80 samples of grapes

State of AF after 18 days at 20°C.

50% : AF< 20%

42.5% : AF between 20 & 50%

7.5% : AF> 50%


Micro-organisms on (healthy) grapesGrapes 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

Metagenomic survey

on merlot grapes

(according to Bauquis,

2017)

Molds and yeast

Yeast diversity in must :

a double component

Flora present on

winery equipment:

Saccharomyces

Candida

Brettanomyces

Grape flora at

maturity

Kloeckera

Hanseniaspora

Candida

Pichia

S. cerevisiae

Metchnikowia

Often majority

(>70%)

Variable levels of population:

103 to 106 cell/ mL

?

Evolution of yeasts floras

According to Blondin, 18 mars 2011, matinée technique des œnologues (Montpellier SupAgro )

Vine Must, beginning

of AF

Wine during AF

>6% alcohol

End of AF, wines

Resistant to SO2 and alcohol

Acetic acid producing yeast

The specific case

of cold soak

Growth of Hanseniaspora uvarum 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 Hanseniaspora uvarum 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 than Saccharomyces.

The specific case of

cold soak

Impact of temperature

• Low temperatures can promote non-Saccharomyces(but also Saccharomyces uvarum).

• At low temperature (15°C), apiculated yeast resistbetter to alcohol. There are cases of dominance of apiculated yeast at the end of AF !

• To reason together with the level of SO2.

• On the opposite, high temperatures (28°C) promote S. cerevisiae.


• Potential production of metabolites of interest:

– Specific fruity esters

– Varietal thiols

– Other aromas…

– Glycerol

• Specific fermentative capacities of some non Saccharomyces strains (osmotolerance, cryophily…)

Yeast diversity in fermentation:

benefits

Aromatic complexity


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:


dangers


ethylacetate

• Potentially high production of SO2 and/or

acetaldehyde

Potential of production of SO2 by

yeast

0

5

10

15

20

25

30

35

40

45

RA17 S1 S7 S24 S25 S34

mg

/LTotal SO2 after AF

Variability of production of

acetaldehyde

A B C D E F G H

Yeast strainAccording to Cheraiti et al, 2010


dangers


ethylacetate


acetaldehyde

• Possible production of volatile phenols

(Brettanomyces bruxellensis, Pichia guillermondi)


dangers


ethylacetate


acetaldehyde

• Possible production of volatile phenols

(Brettanomyces bruxellensis, Pichia guillermondi)

• Negative interactions with S. cerevisiae

Consommation de la thiamine par K. apiculata

T i m e ( h )

0 1 2 3

[14C

-Thia

min

] (µ

g l

-1)

0

1 0

2 0

3 0

4 0

5 0

6 0

And bacteria ?

• Sometimes, stronger contaminations

And bacteria ?

0

0,1

0,2

0,3

0,4

0,5

0,6

0,7

0,8

10°C 16°C 10°C 16°C

Acetobacter Gluconobacter

Ace

tic

acid

(g

/L)

Production of acetic acid after contamination of the must with acetic bacteriaPasteurized 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

T0

T 7 days

T 14 days

T 21 days

Fermentation with local flora :

balance benefits/risks

Microbiological risks and sulphiting

Prefermentative microbiological

risks:

• Kloeckera apiculata

(Hanseniaspora uvarum)

• Brettanomyces bruxellensis

• Some bacterial risks

• AF triggering by indigeneous

S. cerevisiae

Settling of the

must

Racking off

Alcoholic fermentation

Beginning AF

1/3 AF

Grapes : grape-gondola or

harvesting machine

End AF

Ageing and storage

Bottling

Skin maceration

or cold soak

Pressing

MLF

PRE-FERMENTATIVE

BIOCONTROL

Metschnikowia fructicola GaïaTM

A first approach:

split addition of yeast

1,00E+00

1,00E+01

1,00E+02

1,00E+03

1,00E+04

1,00E+05

1,00E+06

Early inoculation (20 g/hL at thefilling of the tank)

Split inoculation (5 g/hL at thefilling then 15 g/hL after cold

soak)

Late inoculation (20 g/hL aftercold soak)

Po

pu

lati

on

(u

fc/m

L)

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

non Saccharomyces yeasts

M. fructicola and biocontrol :

an old story?

Wound then inoculated apples :

- 1st series with sterile water (10µL)

- 2nd series with a suspension of M. fructicola (5.107 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

Controls

M. fructicola


No fermentative

power

Antimicrobial activity,

especially anti-Kloeckera

Excellent

implantation… and

survival

No production of

undesirable

metabolites

Positive sensory

contributionLow nutritional

needs

M. fructicola (GaïaTM):

A true prefermentative tool

0

20

40

60

80

100

120

0 50 100 150 200 250 300 350

CO

2 (

g/L

)

Time (hours)

Merlot at 14°C from T=0 to 96h, then increase to 24°CMetschnikowia : 25g/hL at T=0, then S. cerevisiae in sequential inoculation at T=96H

Control S. cerevisiae : 25g/hL at T=0

Gaïa

S. cerevisiae

T=96H

Modality Metschnikowia :

S. cerevisiae inoculation

T=0inoculation

Control: S. cerevisiae

Gaïa: Metschnikowia

Raynal et al, 2014


No fermentative

power



Excellent

implantation… and

survival

No production of

undesirable

metabolites

Positive sensory


needs

Implantation and survival

of M. fructicola GaïaTM

• On must at low temperature and on long-term

1,0E+00

1,0E+01

1,0E+02

1,0E+03

1,0E+04

1,0E+05

1,0E+06

1,0E+07

1,0E+08

0,0 0,1 12,0 30,0 40,0 50,0 60,0 80,0

Time (days)

Populations of Metschnikowia (cfu/mL) depending on the inoculation temperature– from 0 to 80 days (muscat stored at 0°C)

0°C

5°C

10°C


No fermentative

power



Excellent

implantation… and

survival

No production of

undesirable

metabolites

Positive sensory


needs

M. fructicola (GaïaTM):

biocontrol against Kloeckera and

volatile acidity

Gerbaux et al, 2015

0,00

0,10

0,20

0,30

0,40

0,50

0,60

0,70

0,80

Non contaminated control Contamination with Kloeckera* Contamination with Kloeckera +biocontrol with Gaïa

Ace

tic

acid

(g

/L)

Production of acetic acid by Kloeckera apiculata wih or without GaïaTM in a must (sugars 230 g/l, pH3.20, no SO2, pasteurization) - (SD: 0,05 g/l) – values given after AF


No fermentative

power



Excellent

implantation… and

survival

No production of

undesirable

metabolites

Positive sensory


needs

M. fructicola (GaïaTM):biocontrol and sensory contribution

in cold soak

4,0

4,5

5,0

5,5

6,0Fruits Intensity

Acidity / RoundnessBalance

Global quality

Sensory evaluation at the end of ageing of a red wine (pinot noir) in tanks of 2,5 hL with cold soak, with o without Metschnikowia - Average values on 2

years.

Gaïa

Metschnikowia B

Control

Gaïa: a tool of biocontrol

against Botrytis cinerea

• B. cinerea: contaminating agent on desiccated

grapes

• Gaïa fights actively against it :

– Production of pulcherriminic acidS. cerevisiae


Preservation of desiccated grapes (raisining)

Competitive asset

• Goal: to limit the post-harvest

growth of Botrytis cinerea, for

desiccated grapes.


Preservation of desiccated grapes (raisining)

Competitive asset

Gaïa 50 g/ ql: 41 days of desiccation

IOC BE YEAST: FERMENTATIVE

BIOCONTROL

Management of SO2 and SO2-binding compounds

IOC BE yeast : interest

• Guaranteeing a tool for controlling SO2 levels in wines:

– By zero production of SO2, and independently of the conditions

– By a very low production of acetaldehyde, which combines SO2

• Consequences:

– “clean” wines

How does yeast work with

sulfates and sulphites ?

Identification of a low (not)

SO2 / acetaldehyde / -

producing

strain

High sulphite-producing strain Low sulphite-producing strain

SO42-

SO2

Homocysteine

Homoserine

SO42-

SO2

Homocysteine

Homoserine

Increased metabolism if:

- High NH4+

- Low temperature

- Presence of sulfates

- Addition of sulphites in must

Birth of a tool to

decrease sulphites in

wine

�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 SO2 whatever the external

conditions are.

« Low SO2 » mother-yeastYeast of enological

interest

Breeding Enhancement

Zero production of SO2

whatever the conditions are

-20

-10

0

10

20

30

40

50

60

Reference yeast A Other reference yeasts(* = additional yeast not included in the

trial)

IOC BE THIOLS

Dif

fere

nce

s b

etw

een

ad

ded

SO

2 an

d t

ota

l SO

2 an

alys

ed in

fin

ish

ed w

ine

(pp

m)

Production of SO2 : differences between added SO2 and total SO2 found in wine

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)

* *

Low production of acetaldehyde

– less bound SO2

-70%

-60%

-50%

-40%

-30%

-20%

-10%

0%

Chardonnay Maccabeu Grenache/cinsault

IOC BE FRUITS: decrasing the acetaldehyde content(deviation between concentrations obtained with IOC BE FRUITS and those ones obtained with reference

yeast

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)).

Amounts in reduced, oxidized and total glutathione

of different inactivated yeast naturally rich in

glutathione.

Kritzinger et al., 2012

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.

Produit 5

Alternatives to lees to protect

wines against oxidation

0,00

0,50

1,00

1,50

2,00

2,50

3,00

sauvignon 2014 chardonnay 2014

Co

nce

ntr

acio

n (

mg

/L)

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)

GLUTAROM EXTRA Control

A better resistance to air exposure

0,000

0,100

0,200

0,300

0,400

0,500

0,600

0,700

0 10 20 30 40 50 60 70 80 90 100

DO

420

nm

Time of air exposure (hours)

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

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 SO2 addition (added at the settling of the must then post AF).

• These results are obtained with an addition at the beginning of AF.

Glutarom

Thanks for your

attention !

Documents

REDUCING SULPHITES CONTENT IN WINES · 2018-09-03 · Vintage 2013 Hand harvest (nearly 20 kgs) Healthy grapes from “organic” parcels (Pinot noir x2, Chardonnay and Sauvignon)