Co-inoculation and w

ine

Chr. Hansen Ferm

entation M

anagement

Services & Products

A definition of ‘co-inoculation’

‘Co-inocu

lation’is the term

used in w

inemaking w

hen…

yeasts (used to m

anage alcoholic ferm

entations -AF)

and

malolactic bacteria (used to m

anage m

alolactic

ferm

entations -MLF)

…are inocu

lated in a different way than the classical and

conve

ntional protoco

l known as ‘sequential inocu

lation’

where:

yeasts are first used for AF m

anagement

and then -when AF is finished-bacteria for MLF

management are inocu

lated into the w

ine

Therefore during ‘co

-inocu

lation’populations of both active

yeasts and m

alolactic bacteria co-exist in m

usts. Each

population of microorganism ach

ieving its ferm

entation.

3 realities behind ‘co-inoculation’

Depending m

alolactic bacteria tim

ing of inocu

lation,

3 different types of ‘co-inocu

lation’have

been identified

yeast

bacteria

Early

co-inocu

lation

Late

co-inocu

lation

Reverse

inocu

lation

Tim

e

yeast

bacteria

bacteria

yeast

Populations peak

at the same tim

e

Populations DO NOT peak

at the same tim

e

Populations DO NOT peak

at the same tim

e

1 –

4 d

ay

sd

ep

en

din

g A

F/M

LF

kin

eti

cs

Co-inoculation: current knowledge

Commonly practiced in some countries:

Australia, France

, Sp

ain on red and/or rosé

wines

France

, Germ

any and South Africa on w

hite w

ines

Experiments have

started in the 80´s and since

2005 are booming

Therefore current knowledge is sufficient enough

today to:

Get an ove

rview of co

-inoculation benefits

benefits

Get a clear picture of co

-inocu

lation risks

risks

Get the elements to define the ‘‘comfort zone

comfort zone’’where

co-inocu

lation is possible and risks are lim

ited

Select the appropriate protocol

appropriate protocol(early, late, reve

rse)

Follow the two ferm

entations through the right parameters to

monitor

monitorthe co-inocu

lation and succe

ed

Co-inoculation benefits

see t

he ‘

ca

se s

tud

y’

pa

rt t

o g

et

qua

nti

tati

ve i

mp

act

Save tim

e

Save energy

Save fruitiness

Control indigenous

population

IMP

AC

T o

n W

INE

RIE

SIM

PA

CT

on

WIN

ER

IES

IMP

AC

T o

n W

INE

SIM

PA

CT

on

WIN

ES

•Reduce

tim

e to m

arket/

respect deadlines

•Im

prove

tank m

anage

ment

•Sa

ve costs of heating

•Reduce

Carbon footprint

•Avo

id sluggish or stuck

ferm

ent

•Avo

id downgrading

•Diace

tyl produce

d by

bacteria is degraded by

yeasts => fruitiness is kept

when needed + w

ine is stabilized

•Keep the initial quality

potential of the w

ine

•Avo

id spoilage

microorganisms

(Brett. & others)

Co-inoculation risks

Increase of

Volatile Acidity

IMP

AC

T o

n W

INE

RIE

SIM

PA

CT

on

WIN

ER

IES

IMP

AC

T o

n W

INE

SIM

PA

CT

on

WIN

ES

•Cost of downgrading

•Additional costs to treat

wines / m

anage

AF &

MLF

•Potential increase of ace

tic

acid giving vinega

r character

Stuck

ferm

entations

Absence of classical

MLF flavors

•Stuck AF due to bacterial

antagonist compounds/

competition for nutrients

•Stuck M

LF due to SO

2

production by ye

asts and/or

long chain fatty acids

•Sp

oilage

microorganisms

(Brett. & others)

•Absence

/ reduction of

buttery, creamy flavo

rs coming

from M

LF ferm

entation

Co-inoculation benefits/risks management

Experiments, trials and scientific literature

help to understand Oenococcus oenimetabolism

in m

ust &

wine

and therefore a frame w

ork to get safe co-inocu

lation

Experiments, trials and scientific literature

help to understand Oenococcus oenimetabolism

in m

ust &

wine

and therefore a frame w

ork to get safe co-inocu

lation

A ‘co

mfort zone’where co-inocu

lation benefits can be reach

ed

with m

inim

um risks has been defined

A ‘co

mfort zone’where co-inocu

lation benefits can be reach

ed

with m

inim

um risks has been defined

O. oeniprefers M

alic Acid ove

r sugars

The ‘comfort zone’for co-inoculation: pH<3.5

(wit

h O

en

oco

ccu

s o

en

i D

VS s

tart

er

cult

ure

s)

2.9

3.5

4.0

O. oeniprefers sugars ove

r Malic Acid

Avoid co-inocu

lation

Co

nsi

der

co

-in

ocula

tio

n

Metabolism

of

Oen

oco

ccus

oen

iin grape juice/m

ust w

ith pH>3.5

Gluco

se/fructose

→D-lactic acid

ace

tic acid (VA)

ethanol

CO

2

Fructose

→Mannitol

L-m

alic acid

→L-lactic acid

CO

2

Metabolism

of

Oen

oco

ccus

oen

iin grape juice/m

ust w

ith pH<3.5

Same Metabolism

than in w

ine

Same Metabolism

than in w

ine

L-m

alic acid

→L-lactic acid

CO

2

Citric acid

→Diace

tyl

2,3-butanediol

ace

toin

pH

O. oeniprefers M

alic Acid ove

r sugars

2.9

3.5

LOW

1 –2 g/L

HIGH

3.2 –5 g/L

Co

nsi

der

co

-in

ocula

tio

n

MEDIUM

2.1 –3.2 g/L

Decision tool and ‘co-inoculation’protocol

MONITORING

-AF until wine is dry and to avo

id sluggish ferm

ent

-Inocu

late DVS below 1020 -1010 in density

-Control Temp. < 25°C

-Follow D-Lactic Acid

MONITORING

-AF to avo

id stuck ferm

ent

-Control Temp. < 25°C

-Follow D-Lactic Acid

3.2

Late co-inocu

lation

O. oeniwill consume

Malic Acid quickly

Therefore if AF is not

finished at the end of

MLF, O. oeni will

degrade suga

rs

and VA w

ill increase

Reverse inocu

lation

O. oeniwill consume M

alic Acid

while yeasts consume sugars

MONITORING

-AF to avo

id stuck ferm

ent

-Control Temp. < 25°C

-Follow D-Lactic Acid

Early co-inocu

lation

O. oeniwill consume M

alic Acid

while yeasts consume sugars

Malic Acid

pH

Co-inoculation: Go/ No Go checklist

YES/ NO

YES / NO

Yeast and DVS have

to be compatible and both of

them have

to produce

low amount of VA

Preferably use Viniflora

®ye

asts and DVS

Yeast strain M

LF compatible

(produce

low leve

ls of SO

2)

Yeast strain producing low VA

YES/ NO

Optimum DVS co

nditions are in the range

20-25°C

Temperature < 25°C

YES/ NO

Above

3.5, O. oeniwill degrade sugars

pH< 3.5

YES/ NO

To ensure a good survival after inocu

lation, SO

2

leve

l has to be as close as possible to 0 ppm

Total SO

2< 20 ppm

@ DVS inocu

lation tim

e

YES/ NO

Stuck

of sluggish AF could lead to deviations

If unkn

own start w

ith at least one sequential

History of good AF

YES/ NO

Heating and cooling are nece

ssary to get safe co-

inocu

lation

Temperature controlled tanks

YES/ NO

Hyg

iene is alw

ays a key factor success

Healthy grapes

Clean equipments &

cellar

1 ‘NO’or more? => Prefer sequential inocu

lation

Prefer sequential inocu

lation

Only ‘YES’? => Select now the appropriate co

Select now the appropriate co-- inocu

lation protoco

l inocu

lation protoco

l

Co-inoculation: relevant protocol checklist

2.9

3.5

1 –3.2

g/L

3.2 –5

g/L

3.2

Re

ve

rse

in

ocula

tio

nR

eve

rse

in

ocula

tio

nE

arl

y c

o-i

no

cula

tio

nE

arl

y c

o-i

no

cula

tio

n

La

te c

o-i

no

cula

tio

nLa

te c

o-i

no

cula

tio

n

Malic Acid

pH

Parameters to follow for a good m

onitoring

Alcoholic ferm

entation

Density (specific gravity)

Falling down from 1100 => 990

Using a densimeter

Temperature

Co-inocu

lation T< 25°C

Using therm

ometer allowed in

food production

Tim

eIn days

Malolactic ferm

entation

Population after inocu

lation

in cfu/ml

Magic number: 106cfu/ml

Using PCR

L-M

alic Acid

Using standard m

ethods

D-Lactic acid production

Using Enzymatic kit

Temperature

Co-inocu

lation T< 25°C

Using therm

ometer allowed in

food production

Viniflora

®range of products and co-inoculation

1 –3.2

g/L

3.2 –5

g/L

Reve

rse inocu

lation

Reve

rse inocu

lation

Early co-inocu

lation

Early co-inocu

lation

Late co-inocu

lation

Late co-inocu

lation

Viniflora

®

CH35

Viniflora

®

CH11

Viniflora

®

CH16

Viniflora

®

CiNe

Viniflora

®

CH11

Viniflora

®

CH35

Viniflora

®

CH16

Viniflora

®

CH35

Viniflora

®

CH11

Viniflora

®

CiNe

2.9

3.5

3.2

3.1

3.4

3.0

3.3

MalicAcid

pH

1 –3.2

g/L

3.2 –5

g/L

Reve

rse inocu

lation

Reve

rse inocu

lation

Early co-inocu

lation

Early co-inocu

lation

Late co-inocu

lation

Late co-inocu

lation

Viniflora

®

CH35

Viniflora

®

CH11

Viniflora

®

CH16

Viniflora

®

CiNe

Viniflora

®

CH11

Viniflora

®

CH35

Viniflora

®

CH16

Viniflora

®

CH35

Viniflora

®

CH11

Viniflora

®

CiNe

2.9

3.5

3.2

3.1

3.4

3.0

3.3

MalicAcid

pH

MalicAcid

pH

pH

Ba

cte

ria

MERIT.ferm

MERIT.ferm

MELODY.nsac

HARMONY.nsac

MERIT.ferm

MERIT.ferm

MERIT.ferm

MELODY.nsac

HARMONY.nsac

MERIT.ferm

MELODY.nsac

HARMONY.nsac

Yea

sts

Conclusion

Co-inocu

lation in w

inemaking is still in the learning phase

Howeve

r trials conducted all around the w

orld for seve

ral ye

ars give enough

inform

ation to define clear routines for successful co

-inocu

lations

‘Co-inocu

lation’represent 3 typ

es of protoco

l:Reve

rse inocu

lation, early co

-inocu

lation and late co-inocu

lation

Using pH and M

alic Acid content of wines it is possible to define:

A ‘co

mfort zone’where risks are m

inim

um: pH < 3.5

Decision tools to select the m

ost suitable protoco

l to use depending initial malic acid

conce

ntration

Both AF and M

LF have

to be carefully monitored to reduce risks (m

ain one is

an increase of VA related to sugar co

nsumption by inocu

lated M

L bacteria)

Chr. Hansen provides:

a full range of ye

asts and m

alolactic cultures suitable for co

-inocu

lation

Services (advice

, decision tools, follow-up) to m

anage co-inocu

lation in

winemaking and get its full benefits:

time &

energy savings,

microbiological stability,

better bio-safety (no biogenic amines production during M

LF…)

Examples of co-inoculation

4 c

ase

stu

die

s

Case 1: Red w

ine from Tempranillo, Spain

Late co-inocu

lation

Late co-inocu

lation

pH: 3.5

Initial Malic Acid: 2.8 g/L

Viniflora

®

CH16

MERIT.ferm

MELODY.nsac

HARMONY.nsac

Protocol

Products

Wine parameters

The rate of malolactic ferm

entation

compared w

ith co-inoculation w

ith freeze

dried Viniflora

®(pH 3.5 green and pH 3.8

yellow) and sequential inoculation (pH 3.5).

Alcoholic ferm

entation w

as completed on day

7, so in this case the co-inocu

lated M

LF

completed at the same tim

e as AF. The

sequential was inocu

lated on day 7, and

completed on day 30, hence, a saving of 23

days. Calculate the Return On Investment

Calculate the Return On Investment

corresponding w

ith

corresponding w

ith Vinisav

Vinisav-- ee

Case 2: white w

ine Pinot Gris, Germ

any

pH: 3.1

Initial Malic Acid: 4.2 g/L

MERIT.ferm

Protocol

Products

Calculate the Return On Investment

Calculate the Return On Investment

corresponding w

ith

corresponding w

ith Vinisav

Vinisav-- ee

Wine parameters

Reverse inocu

lation

Reverse inocu

lation

Viniflora

®

CH11

Chr. Hansen had significant succe

ss w

ith co-inocu

lating

Viniflora®

CH11 (FroZen™ve

rsion) cultures in

Sparkling base w

ines and low pH w

hite w

ines, notoriously

difficult to induce

MLF.

In Germ

any, the m

alolactic culture w

as inocu

lated 4 days

before the yeast w

as inocu

lated, representing a ‘reve

rse’

inoculation. After 4 days, 0.5 g/L of malic acid w

as

already conve

rted to lactic acid, before the onset of AF.

Malolactic ferm

entation w

as completed 25 days after

inoculation.

Important: Total SO

2in the juice w

as less than 20 ppm.

.

Case 3: Chardonnay sparkling base, South Africa

pH: 3.2

Initial Malic Acid: 4.0 g/L

MERIT.ferm

Protocol

Products

Calculate the Return On Investment

Calculate the Return On Investment

corresponding w

ith

corresponding w

ith Vinisav

Vinisav-- ee

Viniflora

®

CH11

In South Africa, Chardonnay was inocu

lated w

ith Viniflora

®

CH11 (FroZen™

version) 24h after ye

ast inocu

lation and

the M

LF completed 14 days after AF.

Norm

ally, the w

inery depends on spontaneous MLF

that can take

betw

een 2-4 m

onths.

The sequential inocu

lated CH11 completed m

ore than

a m

onth after inoculation.

Important: Total SO

2in the juice w

as less than 20 ppm.

Wine parameters

Early co-inocu

lation

Early co-inocu

lation

Case 4: red w

ine Gamay, France

pH: 3.4

Initial Malic Acid: 3.0 g/L

MERIT.ferm

Protocol

Products

Calculate the Return On Investment

Calculate the Return On Investment

corresponding w

ith

corresponding w

ith Vinisav

Vinisav-- ee

Viniflora

®

Oenos®

In Southern Burgundy region of France

a very fast proce

ss

is required in order to produce

Beaujolais Nouve

au. Early

co-inocu

lation suits this perfectly.

After an initial therm

o-vinificationstage

, Viniflora

®Oenos

(FroZen™

version) is inocu

lated into the ferm

enting m

ust,

24h after the yeast is added at pressing. The M

LF

completed 14 days after AF.

Both ferm

entations being complete in two w

eeks meets

both m

arket requirements and appellation rules

Wine parameters

Early co-inocu

lation

Early co-inocu

lation

Thank you for your attention!

Get the latest update about

ferm

entations management on our web site:

www.chr-hansen.com/w

ine