The utilization of non-Saccharomyces Yeast for organoleptic properties and Bioprotection -- Chr-Hansen Hentie Swiegers Ad van Etten Viniflora® Non-Saccharomyces yeasts Bio-science to protect & create -- 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 -- 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 -- 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