A novel malolactic strains “Viniflora Sparta” with high acidic resistance improved the aroma profile in Orion white wine. Carl-Henrik Brogren1,2 and Nicolai Staugaard Hansen3. Bernstorff Castle Fruit and Wine Corporation1, Bernstorff Slotshave, Ved Slotshaven 3, DK-2820 Gentofte and VinoSigns.dk2, The First Scandinavian Enology Laboratory, Henningsens Alle 38, DK-2900 Hellerup, Denmark. & Dept. of Wine and Fermented Beverages3, International Operation, Christian Hansen A/S, Boege Alle 10-12, DK-2970 Hoersholm, Denmark. henrik@brogren.dk or nicolaistaugaard@gmail.com

Wine-style balance range (ref: WineMaker April/May 2017 p.36) 01/08/2018 Carl-Henrik Brogren - VitiNord 2018 - Malmø/Copenhagen

Grape Malic acid content decreases during the ripening period http://www.bcawa.ca/winemaking/acidph.htm Regulation of malate metabolism in grape berry and other developing fruits. Sweetman C, Deluc LG, Cramer GR, Ford CM, Soole KL. Phytochemistry. 2009 Jul-Aug;70(11-12):1329-44. Véraison -----------------------Ripening ---------------------- Harvest 01/08/2018 Carl-Henrik Brogren - VitiNord 2018 - Malmø/Copenhagen

Carl-Henrik Brogren - VitiNord 2018 - Malmø/Copenhagen Malolactic ”fermentation” is an enzymatic decarboxylation which reduces acidity and removes Malic acid bitterness and astringency. Under anaerobic condition (minus O2) Under aerobic condition (plus O2) The proces of MLF is carried out by LAB (e.g. Oenococcus oeni) and involves the conversion of malic acid to lactic acid. 01/08/2018 Carl-Henrik Brogren - VitiNord 2018 - Malmø/Copenhagen

Carl-Henrik Brogren - VitiNord 2018 - Malmø/Copenhagen Malolactic ”fermentation” – its additional organoleptic benefits on the sensoric properties of the wines Malic acid removal (partial or complete) – lower astringency – a harsh tasting acid. Formation of Lactic acid – with a softer tasting acid, which enhances mouthfeel. Increasing the pH – lowering acidity by removing one carboxylic group. Induces more diacetyl formation – with a buttery flavor (Chardonnay like). Induces new flavor components - like higher alcoholes, aldehydes, esters, lactones, and new acids, etc. Produces glycosidase enzymatic activity – which cleaves glycosides (sugar- conjugates) and thereby releases new important flavor components. 01/08/2018 Carl-Henrik Brogren - VitiNord 2018 - Malmø/Copenhagen

Previous comparative studies of malolactic strains Viniflora Oenococcus Oeni cultures with difference acidity robustness used for malolactic fermentation Previous comparative studies of malolactic strains 01/08/2018 Carl-Henrik Brogren - VitiNord 2018 - Malmø/Copenhagen

Carl-Henrik Brogren - VitiNord 2018 - Malmø/Copenhagen A comparative kinetic study with sequential MLF on Orion white wine (pH=2.9) using Viniflora CH-11 and Viniflora Sparta (Chr-Hansen) 01/08/2018 Carl-Henrik Brogren - VitiNord 2018 - Malmø/Copenhagen

Acidic contents in Solaris and Orion juices and wines from 2017 before/after MLF with or without deacidification. SOLARIS 2017 – 11% alc ORION 2017- 11% alc GENERAL RANGE Grape juice White wine before MLF White wine after MLF (Vinoferm Malocid) after deacidification White wine before MLF White wine after MLF (Viniflora CH11) White wine after MLF (Viniflora Sparta) acidity Grape wine acidity Tartaric Acid (gram/L) 5.7  5.4  4.2  5.9 5.5  4.3  4.1  5-6  4-6  Malic Acid (gram/L) 3.8  3.4  0.18 (-95%)  3.9  1.9 (-51%)  0.06 (-98%)  3-10 2-5 (-MLF) <3 (+MLF)  Total Acid (gram/L as tartaric acid 10.8 10.2  6.3  12.2 10.8  8.2  6.2  9-11 (fruity) 7-9 (mature)  8-9 (fresh) 6-8 (bold)  pH 2.8 3.0 3.3 2.7 2.9  3.4 2.8-3.4 3.1-3.5 01/08/2018 Carl-Henrik Brogren - VitiNord 2018 - Malmø/Copenhagen

Sensoric judgement after MLF treatment An panel of 8 persons at the Dept. of Wine and Fermented Beverages blind tasted the 3 white wines. All taster were impressed by the level and cleaness of the vinification. 4 persons gave the highest score to the Orion MLF “CH-11” wine, and the 4 other persons scored the Orion MLF “Sparta” wine highest. All could taste the higher acid level in half malolactic “CH-11” treated wine. In comparison. the Solaris “Malocid” MLF treated wine had - as expected - a more intense flavor with more thiols and elderflower and tropical aromas. Some “mousiness” was detected in the Solaris wine, probably since half of our Solaris harvest in 2017 was Botrytis infected, why even the uninfected grapes might carry over some. The Botrytis infected Solaris grapes were cryopressed and used for a Sauternes production. 01/08/2018 Carl-Henrik Brogren - VitiNord 2018 - Malmø/Copenhagen

Carl-Henrik Brogren - VitiNord 2018 - Malmø/Copenhagen Conclusions With a few exceptions, MLF is recommended for cool climate white wine and sparkling wine production. Malic Acid content decreases drastically during the ripening period. MLF can removes partly or fully the unwanted high level of Malic acid in not fully matured grapes. MLF lower the extensive acidity in many cool climate harvested grapes and remove unwanted bitterness, MLF one white wine below pH 3.2 needs specific selected strains of malolactic (Oenococcus Oeno) strains with especially adapted high acidity resistance. Viniflora CH-11 and the ”novel” Viniflora Sparta can be used to initiated MLF in grape juice with acidity below pH 3.0, without any need for preceeding deacidification, e.g. with calcium carbonate. Alternative methods to remove Malic acid: Use special recombinant maloalcoholic yeast strains, like ProMalic (Lallemand), ML-01 (Springer-Oenologie), or non-recombinant Lalvin71B (Lallemand) 01/08/2018 Carl-Henrik Brogren - VitiNord 2018 - Malmø/Copenhagen

Thanks for your attention! Easy reading about MLF: Mike Miller: Wine from the Inside Out – Malolactic Fermentation (e-Book 2) https://www.yumpu.com/en/document/view/20192477/e-book-2-malolactic-fermentation-accuvin-wine-tests-home-page Contact: henrik@brogren.dk or nicolaistaugaard@gmail.com 01/08/2018 Carl-Henrik Brogren - VitiNord 2018 - Malmø/Copenhagen

Carl-Henrik Brogren - VitiNord 2018 - Malmø/Copenhagen WINE ANALYSIS FOSS WineScan analytical data versus Randox enzymatic data and Accuvin quick-tests data. Test system Wine Malolactic strain Total acids ( g/L) Tartaric acid Malic acid Lactic acid Volatil acids Acidity (pH) Ethanol (%Vol Alc.) Citric acid Glycerol ( g/L) Winescan Solaris Malocid 5,61 (9,8) 4,1 0,18 (3,75) 1,81 0.26 3,3 (3,0) 11,1 0,41 4,2 Orion 1 CH-11 7,03 (9,6) 4,3 1,85 (3,45) 1,16 0,26 3,1 (2,9) 10,8 0,39 4.5 Orion 2 Sparta 5,95 (9,6) 0,16 (3,45) 1,97 0,27 3,2 (2,9) 10,6 Randox 4,15 0.23 1.99 0,32 11.3 4.1 4.34 1.97 1,25 0.30 11.1 4.4 4,32 0.19 2.12 0,33 Accuvin 6 (11) 2 3,4 11 8 (10) 1 3,2 7 (10) 3,3 01/08/2018 Carl-Henrik Brogren - VitiNord 2018 - Malmø/Copenhagen

Malolactic fermentation – effects on pH and total acidity 2.9-3.0 3.1-3.3 01/08/2018 Carl-Henrik Brogren - VitiNord 2018 - Malmø/Copenhagen

VitiNord 2018 – submitted Abstract A novel malolactic strains “Viniflora Sparta” with high acidic resistance improved the aroma profile in Orion white wine   Carl-Henrik Brogren and Nicolai Staugaard Hansen* Bernstorff Fruit and Wine Corporation, Ved Slotshaven 3, DK-2820 Gentofte and VinoSigns.dk1, Henningsens Alle 38, DK-2900 Hellerup, Denmark, and *Dept. of Wine and Fermented Beverages, International Operation, Christian Hansen A/S, Boege Alle 10-12, DK-2970 Hoersholm, Denmark henrik@brogren.dk or *nicolaistaugaard@gmail.com In cool climate regions grape maturation is often delayed and incomplete with a high level of malic acid. This lowers the pH-level in the juice, so either malolactic fermentation or de-acidification are needed. The drawback of de-acidification is removal of tartaric acid, while malolactic fermentation below pH=3.2 often is not successful. The aim of this study is to demonstrate that a novel strain of Oenococcus Oeni, Viniflora® SpartaTM, having a high tolerance to low pH, can initiate malolactic fermentation at pH≥2.9.  The study was performed on Orion grape juice harvested in Denmark, October 2017, in one of the coldest and most humid autumns. Two equal aliquots of 25L freshly destemmed and hydro-pressed Orion juice were fermented into white wine for 4 weeks. No de-acidification but a slight chaptalization was done. The final wine had a pH=2.9, too acidic for a standard malolactic fermentation. Four weeks after ended alcoholic fermentation, one batch was added Viniflora® CH11, acidity resistant at pH≥3.0, the other batch the novel Viniflora® SpartaTM, resistant at pH≥2.9. Samples were collected twice weekly for 3 weeks and analyzed on Winescan (Foss) for the content of malic and lactic acid, plus other parameters. The Viniflora® SpartaTM treatment gave a fast and complete removal of malic acid, while the Viniflora® CH11 strain acted slower and didn't complete malolactic fermentation within the time span of the experiments. For comparison a batch of 25L Solaris grape juice was deacidified to pH>3.2 and malolactic treated with Vinoferm® Malocid, only active above pH=3.1. Sensory scoring of the white wines indicates that an organoleptic improved aroma profile was obtained after complete malolactic treatment with Viniflora® SpartaTM. De- acidification decreased the tartaric acid content with negative impact on the wine aroma. In conclusion, we recommend malolactic fermentation of high acidic immature grape juice harvested from cool climate cultivars. 01/08/2018 Carl-Henrik Brogren - VitiNord 2018 - Malmø/Copenhagen