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