The utilization of non-Saccharomyces

Yeast for organoleptic properties and

Bioprotection

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Chr-Hansen

Hentie Swiegers

Ad van Etten

Viniflora® Non-Saccharomyces yeasts

Bio-science to protect & create

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Chr-Hansen´s expertise is in fermentation management

- 1980 Introduced Viniflora™; first direct inoculation concept for MLF - 2004 First to introduce non-sacch/sacch yeast blends

- 2006 Introduced FroZen direct inoculation MLF cultures a cost efficient solution for large wineries. st - 2009 Prelude™, the 1 pure Torulaspora delbruecki - 2010 Frootzen™, the 1st pure Pichia kluveri st - 2011 Concerto™, the 1 pure strain of Kluyveromyces thermotolerans - 2011 CiNe™, 1st citrate negative malolactic culture

- 2012 Freasy™, a frozen MLF concept that can be kept for 3 months at -18C st - 2014 NOVA™, 1 MLF concept for low/no sulfite wine making process (US approval pending) - 2014 Biggest single customer order for direct inoculation

MLF cultures - 2014 Full innovation pipeline to stay ahead

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MFC* sequence in winemaking

Alcoholic Malolactic

fermentation fermentation

Saccharomyces

spp. Oenococcus oeni / Lactobacillus ssp.

Non-Saccharomyces

spp.

Population

Time

in days then weeks * MFC: Microbial Food Cultures

Bio-science to protect

Where a high concentration  of identified and living microorganisms is used -  instead of preservatives-, to avoid the development of spoilage microorganisms (yeasts, molds, bacteria) that could degrade  product microbial quality, product

organoleptic characteristics and by that spoil or render the product unsafe.

 the use of Saccharomyces cerevisiae starter cultures to kick-off alcoholic fermentation and produce ethanol to reduce the risk of spoilage organisms developing in the must (e.g. spontaneous yeast)

 the inoculation of wine with known DI Oenococcus oeni strains to manage MLF and suppress undesirable Lactic Acid Bacteria like Pediococci

 the use of pure strain Torulaspora delbrueckii at the pre-fermentation maceration phase, outcompeting wild yeasts and or molds

Bio-protection in winemaking:

Competitive exclusion Compounds production

- The culture or the blend of cultures selected can - The culture or the blend of cultures selected are overcome other species/strain of microorganisms able to produce specific compounds limiting the growth in specific conditions development of technical or pathogenic contaminants

- The effect is linked to both: - The effect is strain dependent and can be linked  the strain(s) selected and therefore its to production of: physiology/production method  ethanol

 the concentration of active cells after inoculation  (until a specific limit of action in cfu/ml) organic acids

 the growth rate of this strain  peptides: mycocines, killer factors, bacteriocines

 … Therefore knowledge, expertise in microbiology and production of cultures is a key success factor

Jameson effect Most numerous bacteria if well adapted, Minority population decelerates when inhibit the growth of the other via physical the majority or the total population reaches its interactions maximum (competition for a common limiting resource You can not reach more than 1E+08/1E+09 cfu/g in

or for space?) solid food

Jameson effect

Protective culture Contaminants

(log cfu/g)

Contaminants (molds, yeasts, Bacterial concentration

bacteria) + protective culture

time Simultaneous deceleration

(Lag and µmax similar)

8

Bio-protection to create:

Winery/Process benefits Consumer benefits

Fermentation Grape Potential Cleaner Recipe Brand love management

Minimise risk of defects and - Control AF and MLF Less or no SO2 in winemaking - Consistency (not uniformity) reduce downgrading related to processes to manage timing or in final wines fermentation mismanagement

- Secure biosafety of berries, Less or no preservatives - Sustainability Bring out true wine potential musts or wines to prevent (ascorbic acid/sorbates…) through protection and release contamination of Flavors; Mouth-feel and Color Reduced negative compound - Health production/off flavours Add full ingredient traceability To fermentation processes Biogenic amine control

Build margins Create value

Reduce sulfites in winemaking

Invent new wines for new customers with Viniflora® example of customer activities (Italy targeting Northern Europe)

Low histamine wine

MLF managed with Viniflora® CH16

http://www.e-magin.se/v5/viewer/files/viewer_s.aspx?gKey=jnj06xmr&gInitPage=33 11 Incremental quality improvements

Quality Yesterday Today Tomorrow

biosafety Health

Process control Waste

Speed CO2

Non- Yield control Comm Yeast Temperature MLF Sacch Sustainability

Functionality

Security

Economy Time

Viniflora® Non-Saccharomyces yeasts

A Chr. Hansen concept for Bio-protection & more

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Application history of non-Saccharomyces yeast in the wine

Kluyveromyces thermotolerans (S. veronae) promoted by Brice Rankine in the late 1940’s and onwards (AWRI 173)

Torulaspora delbrueckii (S. rosei) deposited by Rainer Eschenbruck in 1975 and promoted for sweet wines (AWRI 1034)

Blend of K. thermotolerans, T. delbrueckii and S. cerevisiae ADY launched in 2007/2008

Single strain T. delbrueckii ADY launched in 2009

Single strain Pichia kluyveri frozen yeast launched in 2010

Single strain K. thermotolerans ADY launched in 2012

14

Non-Saccharomyces in the ‘wine literature’

The Microbial Population Dynamics in Wine Saccharomyces takes over,

after starting from a low conc.

Non-Sacc’s proliferate at early stages

Goddard MR. 2008. Ecology 89: 2077-2082

16

Yeast Diversity in Wine

The ecology of micro-organisms during winemaking is very

complex. The following species of yeast can be found:

• Brettanomyces / Dekkera • Saccharomycodes

Candida • • Schizosaccharomyces

• Cryptococcus • Zygosaccharomyces

• Debaromyces • Metschnikowia

• Hanseniaspora / Kloeckera • Pichia

• Hansenula • Rhodotorula

• Kluyveromyces

• Torulaspora

17

What properties do non-Saccharomyces yeast develop in wine?

18

Non-Sacch yeasts: wine characteristics impacted per type

Flavors Acid balance Mouth-feel

Precursors conversion Organic acids Polysaccharides Metabolism differences production/reduction Mannoproteins

1 species among wine NSAC yeast 1 species among wine NSAC yeast 1 species among wine NSAC yeast has shown the highest has shown the highest potential: has shown the highest potential: Pichia kluyveri Lachancea* thermotolerans potential: Torulaspora delbrueckii

FrootZen™ Concerto™ Prelude™

* formerly Kluyveromyces thermotolerans 19

Viniflora® FROOTZEN™

FrootZen™: the magic of direct inoculation

Sauvignon blanc, 2010, New Zealand

3000

2500

2000

3MH 1500 3MHA

1000

Volatile thiols (ng / l) 500

0 Control Pk Co-inoc Pk Seq

22

Thiols analysis on Sauvignon blanc

3-MH (µg/l)

340

320 300

280 260 240

220 200 Cuve 24 – control Cuve 25 - Frootzen™

sample 3-MH (µg/l) 3MHA (µg/l)

Cuve 24 – control 240 33

Cuve 25 - Frootzen™ 324 42

Sensory profile improvement with FrootZen™

white wine example (Sauvignon blanc)

Range of flavours mentioned by external trained/non professional jury Loire Valley wine (Sancerre - Sauvignon blanc)

Data: In senso veritas, Feb. 2013

Tropical fruit notes Floral notes

Sensory profile improvement with FrootZen™ red wine example (Pinot Noir)

Viniflora® FrootZen™ : main applications

Fruit forward wines:

Chardonnay, Viognier, Riesling Pinot gris, S. blanc, Chenin…

Pinot Noir, Syrah Merlot, Cabernet-Sauvignon, Grenache N.

Fruit forward rosé wines

Viniflora® PRELUDE™

Torulaspora delbrueckii - effect on palate-weight: high production of polysaccharides

Sc alone Sc + Torulaspora delbrueckii

Source: Comitini, F., et al., Selected non-Saccharomyces wine yeasts in controlled multistarter f..., Food Microbiology (2011)

28 2 June 2015 Australia & NZ, July 2012 Viniflora® Prelude™ - Torulaspora delbrueckii example of volatile acidity reduction & fatty acids reduction

Acetic acid Glycerol Esters Fatty acids (C6,8,10,12) g/l g/l mg/l mg/l

S. cerevisiae (EC1118) 0.21 6.7 6.3 14.5

T. delbrueckii (Prelude™) 0.06 6.9 7.3 3.5 + S. cerevisiae (EC1118) p.t. ~ 20 mg/L

The primary reason for the decrease in volatile acidity and fatty acids is the high tolerance of Td yeasts for high sugar substrates, they are less stressed by their environment than Saccharomyces spp.

29 Sensory effect of Prelude

Flavor intensity* 9 Flavor intensity in mouth* Reduction intensity 8

Herbaceous 7 Defaults*

6

5

Alcohol 4 Vegetable / Pyrazines

3

2

Biterness 1 Green thiol 0

Astringence Fruity thiols*

Acidity Fruity terpens

Sugar Fermentation

Palate weight Mineral

CONTROL PRELUDE + CONTROL

Comparison between control in purple (S. cerevisiae) and experiment in blue (PRELUDE™ + S. cerevisiae) 30 demonstrating the higher flavor intensity with PRELUDE™ in wines from Sauvignon Blanc. Experiment and grading achieved externally, in France by IFV Sud Ouest, V´innopôle 2010 PRELUDE™ : main applications

Woody whites Pinot gris Chardonnay

Merlot, Cabernet-Franc, Cabernet sauvignon Pinot Noir,

Delicate and low alcohol rose

Viniflora® CONCERTO™

Concerto™ - Kluyveromyces thermotolerans – strain CH456 lactic acid production -natural acidification- ideal for warm climates red or rosé wines

Sc only Kluyveromyces* thermotolerans

control + Sc

Source: Comitini, F., et al., Selected non-Saccharomyces wine yeasts in controlled multistarter f..., Food Microbiology (2011)

* : Kluyveromyces thermotolerans is also named Lachancea thermotolerans (L. thermotolerans)

Ethyl lactate synthesis

34

Ethyl lactate production

70

60

mg/l 50

40 lactate

30

Ethyl 20

10

0

S. cerevisiae Prelude Concerto Spontaneous

35

Non Sacch: Sequential Inoculation, up to 48 hours before S.cerevisiae

Specific gravity cfu/ml ° Brix of wine Inoculation of Concerto™ Inoculation with usual standard S. cerevisiae yeast

25-35 points ° 1100 up to 6-8 B 24°B 1E+11

1E+10 1070 17°B 1E+09

Wine ready for 1030 malolactic fermentation 8°B with Viniflora® cultures

990 0

Must Wine Time

36 2 June, 2015 Australia & NZ, July 2012 CONCERTO™ : main winemaking applications

White wines from warm climate areas Chardonnay, Semillon, Chenin

Merlot, Cabernet sauvignon Grenache N., Syrah, Pinot Noir

Rosés wines from warm areas

60% saccharomyces cerevisae

20% torulaspora delbrueckii

20% kluyveromyces thermotolerans

Three in one MELODY™ Chardonnay study by the AWRI

Chemical Analysis

40

Consumer Testing

*Only Segment 1 showed statistically significant differences

** Segment 1 represents regular wine drinkers

41

‘Sensory summary’ of impact of non-Saccharomyces yeast

42

Winemaking:

Sacc: S.G. 101 AF temp: 16°C Maceration: 4 hours MLF: No

Analysis:

RS: 3.5 g/l Alc: 14.0% pH 3.5

TA = 4.8g/l (as tartaric)

43

Cabernet Sauvignon

Acidity 7

Winemaking: Green Beans 6 Fruitiness Sacc: S.G. 101 5 AF temp: 25°C 4 Maceration: 6 days MLF: Yes 3 Honey 2 Body

1 Analysis: RS: 3.0 g/l 0 FrootZen

Alc: 13.8% Prelude pH 3.6 Blackberry Bitterness TA = 4.8g/l Concerto

(as tartaric)

Strawberry Cherry

Black plum

44

Alcohol production by non-Saccharomyces yeast

14

0,5% less 12 ethanol

10 Merit, Sacch, 8 Concerto, Prelude, 6 Alcohol % Melody,

4 Concerto, incr. dosage Prelude, incr. dosage

2

0 0 5 10 15 20

45

Fermentation kinetics of non-Saccharomyces yeast

250

200

g/l Control 150 Merit / Fruc

Concerto

Gluc 100 Concerto incr. inoc.

Prelude 50 Prelude incr. inoc.

0

Day Day Day Day Day Day Day Day Day

0 2 5 7 11 14 19 22 30 46

Viniflora® range of yeasts

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