Multi-Yeast Strategies for Cider Fermentations
Cider aromatic diversity: the origins
Aromatic potential from apples
(apple variety, orchard practices)
Aromas from apples
Enzymatic activities Alcoholic
Fermentation
Losses (oxidation)
Maturation (ageing aromas)
Masks (contaminations, reduction…)
Exchanges (lees, oak)
Global cider aromas
Introduction
3
Aroma compounds
3 types :
Varietal aromas Fermentative
aromas Post-fermentative
aromas
• Liberation of aroma compounds from non-aromatic precursors
• Thiols, terpenes,…
• Aromatic compounds from yeasts metabolism
• Higher alcohols, esters
• Development of aroma during aging of wine
Influence of yeasts Studied aromas
And now… Shea
A dynamic biodiversity
• At the beginning of AF: S. cerevisiae isn’t the main yeast !
• Non-saccharomyces yeast
die off during AF
• There is a niche for a
great diversity of
enzymatic activities (d’après G. Fleet, 1990)
S cerevisiae
Espèces Kloeckera/Hanseniaspora
Espèces Candida
How can we take advantage of this biodiversity ?
Option 1 : delayed yeast-inoculation…
a) Important risk of non implantation >> Stuck AF
b) A lot of non-Saccharomyces should be avoided
c) Population of non-Saccharomyces varies a lot from one vintage to another >> unreliable process.
Delayed yeast-inoculation : spontaneous beginning of AF, then inoculation with a selected S. cerevisiae yeast ?
Selected S. cerevisiae
Option 2: sequential inoculation
How can we take advantage of this biodiversity ?
Biodiversity is under-explored Saccharomyces: 7 species
Saccharomyces cerevisiae
Saccharomyces paradoxus
Saccharomyces pastorianus(syn.carlsbergensis)
Saccharomyces bayanus
Saccharomyces mikatae
Saccharomyces cariocanus
saccharomyces chevalieri
Saccharomyces kudriavzevii
Saccharomyces boulardii(=infra-tipo S.cerevisiae)
Debaryomyces : 15 species including 9 used in food industry
Debaryomyces hansenii (ou candida glabata)
Debaryomyces carsonii
Debaryomyces etchellsii
Debaryomyces maramus
Debaryomyces melissophilus
Debaryomyces robertsiae
Debaryomyces vanrijiae
Debaryomyces polymorphus
Biodiversity is under-explored
Candida: 163 species including around 60 implicated in food
Candida albicans
Candida glabata
Candida guilliermondii
Candida dubliniensis
Candida lusitaniae
Candida parapsilosis
Candida tropicalis
Candida dubliniensis
Candida etchellsii
Candida maltosa
Candida shehatae
Candida milleri
Candida oleophila
Candida pelliculosa
Candida rugosa
Candida sake
Candida utilis(=P.haidinii)
Candida Versatilis
Candida Zelanoydes
candida Intermedia
Candida famata
Candida magnoliae
Etc…
Etc…
Etc…
Biodiversity is under-explored
Lactobacillus plantarum Lactobacillus mali Lactobacillus kefiri Lactobacillus lindneri Lactobacillus brevis Lactobacillus buchneri
Enterococcus faecium Enterococcus avium
Enterococcus hermanniensis Enterococcus durans
Lactobacillus kunkeei Lactococcus lactis
Leuconostoc mesenteroïdes Pediococcus parvulus Pediococcus damnosus
(Oenococcus oeni)
Grape Must
L. plantarum
L. hilgardii ... P. parvulus L. casei L. sanfrancisensis
O. oeni 0-10%
AF
O. oeni 80-100%
L. plantarum P. parvulus L. hilgardii
12
MLF
O. oeni
Aging
(O. oeni)
Lactobacilli Pediococci
Why O. oeni?
(Lactobacilli) (pediococci)
The main LAB species during MLF
Understanding the biodiversity
…in bacteria
Impact of these species
Ester production.
Terpene production.
Hydrolytic enzyme production (release of bound flavor precursors).
Source of stress for Saccharomyces
Ethyl Acetate producers:
– Kloeckera/Hanseniaspora
– Candida stellata
Ester producers, enzyme producers..:
– Candida: otras especies.
– Metschnikowia
– Issatchenkia
– Pichia species
Let’s take advantage of biodiversity!
Sequential inoculation
Aims of the project
- Strains : - Enzymes :
• Glycosidases • β-lyases • Proteases
- Aroma compounds :
• Thiol • Terpene • Higher alcohols
15
• Esters • Organic acids
Liberation of varietal aroma compounds
Liberation of nitrogen sources
Metschnikowia (2 fructicola and 1
pulcherrima) Torulaspora delbrueckii
Saccharomyces cerevisiae
Results
- Metabolites production
16
Strain Sugar
(g/L)
Ethanol
(g/L)
Glycerol
(g/L)
Acetate
(g/L)
Succinate
(g/L)
T. delbrueckii 1 95 7.36 0.10 1.42
M. pulcherrima 128 32 4.20 0.07 0.53
M. fructicola L 110 38 4.35 0.07 0.82
M. fructicola G 115 36 4.74 0.15 0.92
S. cerevisiae 5 95 5.85 0.49 0.65
Main central carbon metabolites - end of the fermentation :
Metschnikowia : Poor sugar consumption
High glycerol yield Little acetate production
Torulaspora : High glycerol and succinate production
Little acetate production
T. delbrueckii production in ADY ? A challenge!
Level2 TD : a new way
• To take advantage of biodiversity in a controlled manner
• New types of aromas – alliance of fresh and dried fruits with pastry notes, with a
low amylic contribution and VA
• Increases mouthfeel
• Increases aromatic length and intensity in mouth
Volatile acidity (g/l H2SO4)
Level²TD291 / Sc complementarity : fermentation properties
Sensorial analysis : increasing mouthfeel
Sequential Inoculation : non-Sacch. and then S. cerevisiae ?
Important change of the aromatic
profile
UMR SPO, Montpellier,
France, Pr. J.M. Salmon
Protocol for cider
! Key-points :
- Rehydration protocol :
temperature is different
(30°C instead of 37°C)
Initial density
(initial °brix)
1st Level :
Torulaspora
delbrueckii
TD291 inoculation
Initial density
– 10/15 points
(initial ° Brix – 1,5/3 °)
2nd Level :
Saccharomyces
Cerevisiae inoculation
Conditions of use : free SO2 < 20 mg/L
! Key-points :
- Respect the timing of inoculation
- Respect the classic rehydration protocol (37°C)
- If necessary, protect the yeast
Just after 2nd Level :
Complement systematically with
complex nutrient (30 g/hL FERMAID O)
Middle of AF :
If necessary, complement
with complex nutrient (depending on initial nitrogen
amount in must and other
limiting factors)
Metschnikowia pulcherrima : interest in Winemaking
Principle: Enzymatic hydrolysis : 2- step sequential reaction:
1.- arabinofuranosidase 2. Β- glucosidase
Free terpenes
release
M. Pulcherrima interesting characteristics: specific
enzymatic pool
Gunata, 1998, A. Ganga,
2008
Flavourless glycosidic complexes are generally more abundant than free
odorous forms and represent an important potential source of wine
aroma.
Level2
A new way for stable sensorial diversity