Rodney S. Hart and Valmary M. Van Breda

Post-Harvest and Agro-Processing Technologies Division,

ARC Infruitec-Nietvoorbij, Private Bag X5026,

Stellenbosch 7599, South Africa

*Email: HartR@arc.agric.za

SOUTH AFRICAN & ITALIAN WINE RESEARCH INNOVATIONS

CURRENT STATUS & FUTURE PROSPECTS

ARC INFRUITEC-NIETVOORBIJ, STELLENBOSCH,

18-19 NOVEMBER 2019

Development of wine yeast – Towards improving wine organoleptic quality

Role of yeasts in winemaking

Van Breda et al., 2013; Pretorius, 2016; Hart et al., 2017

• Wine is an alcoholic beverage produced from the fermentation of grape juice.

• Wine yeast i.e. Saccharomyces cerevisiae.

• Co-inoculation strategies using wild yeast e.g. Torulaspora delbrueckii.

• Grape sugar as carbon-source to produce alcohol & CO2.

• Convert aroma inactive compounds into volatile aroma-enhancing compounds in wine.

• Convert “neutral” juice into aromatic wine.

• Wine yeasts form an integral part of winemaking.

Modulating wine aroma using yeasts• Wine is considered the metabolic footprint of yeast strain.

• Inoculate base must with three (3) different yeast strains.

• Final wines will have different aroma and flavour profiles.

Howell et al., 2006; Mapelli et al., 2008; Pretorius, 2016

ARC Infruitec-Nietvoorbij yeast breeding• South African winemakers are continually striving to increase wine

quality.

• Winemaking styles continually changes - market trends e.g. low alcoholwines, wines with enhanced tropical fruit aromas, etc.

• Climate change is already affecting viticulture and grape physiology(higher Balling).

• Creating new fermentation challenges – low alcohol wines.

• Local and imported yeast strains is available - many fall short.

• High thiol-releasing strain erratically produces undesirable volatile acidity.

• The ARC Infruitec-Nietvoorbij yeast breeding programme comprises ofthree sections, namely,

1. selection of yeasts from nature,

2. breeding of hybrid yeasts, and

3. screening and evaluation of yeasts for suitability for specific winemakingconditions and styles.

• Common goal of the project - increase the number yeast strains availableto winemakers as yeast criteria changes.

Selection of yeasts

White grape cultivars

(varieties) e.g.

Sauvignon blanc

Wine yeast Saccharomuces cerevisiae isolates formpart of the ARC Infruitec-Nietvoorbij yeast culturecollection

White wine grapes e.g. Sauvignon blanc were collectedaseptically from vineyards in Cape Winelands, crushed andallowed to spontaneously ferment until dryness (totalresidual sugar <5 mg/L).

Spontaneous fermentation until dryness(residual sugar <5 g/L)

Single wine yeast Saccharomyces cerevisiae colonieswere randomly selected and characterised by DNAkaryotyping.

Fermented must was subsequently plated ontoYPD agar plates containing 0.1 g/L chloramphenicol

Transfer pure cultures into cryo-tubes containing aglycerol/YPD broth mixture.

Red grape cultivars

(varieties) e.g. Shiraz

Random selection of single S. cerevisiae colonies &characterisation by DNA karyotyping (Carle & Olson,1985).

Fermented must were sampled, plated onto YPD agarcontaining 0.1 g/L chloramphenicol.

Red wine grapes e.g. Shiraz were collected aseptically fromvineyards in Cape Winelands, crushed and allowed tospontaneously ferment until dryness (total residual sugar <5mg/L).

Evaluation of yeast strains

Fermentation potential of isolates are tested in laboratory-scale (0.75 L) fermentation trials

Pilot- ( > 100 L) and semi-commercial scale (≥ 1000 L)fermentation trials, respectively during subsequent vintages

Small-scale (20 L) fermentation trials with promisingisolates during the next vintage

White grape cultivars

(varieties) e.g.

Sauvignon blanc

Red grape cultivars

(varieties) e.g. ShirazLaboratory-scale (0.5 – 5 L) fermentation trials followed by chemical analyses. Pilot-scale (≥ 100 kg) fermentation trials.Small-scale (≥ 20 kg) fermentation trials with promising yeasts.

Semi-commercial scale (≥ 1 000 kg) winemaking trials withADEY.

Gas chromatographic (GC) analyses of wine.OenoFOSSTM - FTIR & Alpha II FTIR Spectrometer(glucose/fructose, ethanol, volatile acid, total acid, malic acid,& pH)

Descriptive sensory analyses of wine.

Nano- LC-MS/MS characterisation of proteinsPilot-scale drying of promising and consistent yeast basedon data, followed by commercialisation.

White & Red grape

cultivars

Breeding of hybrid yeasts

Carle & Olson, 1985; Hart et al., 2019)

Figure: Schematic depiction of inter-genus (inter-genus) hybrid yeast breeding between Saccharomyces cerevisiae and

Torulospora delbrueckii by deploying the protoplast fusion technique

Inter-genus hybrid yeasts

• Saccharomyces cerevisiae x Torulaspora delbrueckii hybrids

MCB C6 M2/1 NT07/1 NT07/2

CHEF analyses of chromosomal DNA of parental yeasts and hybrid yeasts

Van Breda, 2018; Hart, 2018

Hybrid breeding – mass mating

DNA karyotyping of hybrid yeaststrains by Pulsed-Field GelElectrophoresis (PFGE).

Characterisation using MALDI MSBiotyping.

0.0

0.5

1.0

1.5

4x10

Inte

ns. [a

.u.]

4000 6000 8000 10000 12000 14000 16000 18000m/z

NH84

Fermentation potential of hybrids areevaluated in fermentation trials.

aa aa

aa

aa aa

a

a

a

a a

Adaptive evolutionSerial – Erlenmeyer flasks

Sequential batch – Bioreactor

1. Simple2. Cheap

1. Controlled cultivation of yeasts2. Automated empty-refill cycles3. On-line monitoring of

fermentation kinetics e.g. CO2-weigh loss etc.

Bellon et al., 2015; Mans et al., 2018

Commercialy availbale yeast strain from the ARC Infruitec-Nietvoorbij yeast culture collection were be streaked ontodifferent growth medium (YPD, YM & YGC)

Single yeast colonies were inoculated into respective brothmedia (YPD, YM & YGC) and sub-cultivated for threegenerations

CHEF DNA Karyotyping of randomly selected yeast isolatesSDS-PAGE analysis

Adaptive evolution

TotalAcidity

pH

EthanolVolatile Acidity

MalicAcid

Tropical fruit

Floral

Vegetative

-1

-0,75

-0,5

-0,25

0

0,25

0,5

0,75

1

-1 -0,75 -0,5 -0,25 0 0,25 0,5 0,75 1

F2 (

24

.05

%)

F1 (34.40 %)

Variables (axes F1 and F2: 58.45 %)Chemical

Sensory

Commercial yeast_YGC

Commercial yeast_YM

Commercial yeast YPD

-2

-1

0

1

2

-2 -1 0 1 2 3

F2 (

24

.05

%)

F1 (34.40 %)

Observations (axes F1 and F2: 58.45 %)

Multiple factor of analysis (MFA) of sensory parameters of Sauvignon Blanc wines. The yeast VIN7

was cultivated in different growth media (i.e. YM, YPD and YGC) prior to fermenting a standard

Sauvignon Blanc grape must.

Adaptive evolution

• Genome editing - easier to produce yeasts with desired properties.• GM yeast strain i.e. ML01 (Springer Oenologie) was commercialised and is authorized for use

in the USA.• This strain contains two extra genes i.e.

1) malate transporter gene (from Schizosaccharomyces pombe);2) and the malolactic enzyme gene (from Oenococcus oeni).

• Using this yeast strain AF and MLF, normally a two-step process, occur at the same time.• Advantages:

1) Wine processing becomes much faster.2) Less risk of wine spoilage - no delay between AF and the onset of MLF.

Bison, 2004; Schuller & Casal, 2006; Vigentini et al., 2017

Genetic engineering (Genome editing)

Genetic engineering (Genome editing)

Shao et al., 2019

CRISPR/Cas9 - The applications of this novel technology.

Pretorius, 2018

The construction of a 'synthetic' wine strain of Saccharomyces cerevisiae capable of

producing raspberry aroma in Chardonnay. This is the first wine yeast containing a

'synthetic DNA circuit' coding for raspberry ketone formation.

Genetic engineering (Genome editing)

Li et al., 2015; Singh et al., 2015, Pretorius, 2017

Schematic depiction of de novo synthesis of resveratrol from glucose or ethanol using bioengineeredSaccharomyces cerevisiae strain.

Genetic engineering (Genome editing)

Views - GM wine yeast• Australian Wine Research - no GM yeasts will be used in

Australian wine.

• Wines of South Africa & SA Wine Council objected to thecommercial use of GM Yeasts.

• Wine and Spirit Board - illegal to produce GM wine inSouth Africa.

• Consumers in Europe and Asia - rejected GMOs.

• In the USA yeasts are classified as processing agents -need no declaration.

• Large producers of Napa Valley – no intention of usingthe commercial GM strain.

• Polls show that a majority of American consumers wouldprefer to avoid GM wines.

• GM yeasts - wine will be seen as just anothermanufactured beverage.

• Kill the ‘naturalness’ and/or ‘symphonics’ of winemaking.

• Supporters of GM yeast – strains with special propertieswill unlock latent aroma and flavour.

Martenson, 2014; Cummins, 2016, Pretorius, 2016

Concluding remarks

• Bioengineering can address short-comings of commercial yeast strains.

• Genome editing to enhance wine yeast strains is not advised, as it isillegal.

• Cape Winemakers Guild (CWG) and South African Wine IndustryCouncil (SAWIC) is against GMO.

• SA wine industry is too dependent on Europe for exports, which areagainst GM produced foods.

• Therefore, the ARC’s approach of developing new strains using classicalmating is still relevant.

• Contributes in maintaining our green image.

Berrie, 2011

Acknowledgements