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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…)
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