Innovations in Wine Production

Improving English Wine by the Application of Technology and Knowledge Deacidification by Promalic , encapsulated Schizosaccharomyces pombe

Introduction
As a young industry UK wine production is unfettered by history and is at liberty to invest in tools and techniques that allow the production of elegant wines to rival the best of both the old and new world. ProMalic produced by ProEnol is an ideal tool for the UK wine industry allowing superior management of malic acid levels; quicker and more reliable than malolactic fermentation; less aggressive than chemical deacidification and with no risk of removing too much tartaric acid. The adoption of ProMalic for still whites and sparkling base wine should provide improved quality by superior acid management and flavour profile, and better winery efficiency due to the removal of malolactic fermentation.

Acidity Deacidification Saccharomyces cerevisiae Schizosaccharomyces pombe Sequential Inoculation Trials The Future of Deacidification

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Acidity
The cool UK climate commonly leads to the production of high acidity / low pH wines, tartaric and malic acid are the most prevalent.
Pros
Improved wine stability Lower SO2 requirement

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Cons
Acidity is higher than consumer preference High acidity causes unbalanced wines High acidity inhibits malolactic fermentation (MLF)

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Acidity, Pros
Improved wine stability
As acidity increases the environment becomes increasingly toxic to spoilage bacteria such as Acetobacter sp. (Drysdale, Fleet, 1988) and Lactobacillus sp. (Edwards etal, 1999) by reducing the integrity of their cell walls. Lower pH and higher acidity increases the tartrate and protein stability of the wine (Riberau-Gayon, Glories, Duboudieu, 2003)

Lower SO2 requirement

As pH decreases the proportion of free SO2 in the molecular form providing antimicrobial activity is increased and so less SO2 is required to prevent spoilage (Henderson 2009).
Graph1, % of different forms of SO2 over a range pH values adapted from Henderson 2009

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Acidity, Cons
Acidity is higher than consumer preference
English wines tend to have a higher acidity than most other still wines and this may be greater than many consumers will be willing to except as perception of sourness is closely attributable to acidity (Amerine, Roessler, Ough, 1965).

High acidity causes unbalanced wines

Wine should be balanced in both weight and taste, a wine with a high acidity requires body and residual sugar for balance (Rankine, 2004). As modern preferences are for a light, dry style high acidity is difficult to balance.

High acidity inhibits malolactic fermentation (MLF)

Bacteria cultures selected for malolactic fermentation due to superior aromatics and reduced fault commonly have a minimum pH, requirement often above 3.2 and a sensitivity to malic acid above 4g/l (chr-hansen.com, 2011; lallemandwine.com, 2011)

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Deacidification
Grapes contain organoleptic and chemically important levels of organic acids. The two most abundant are tartaric acid and malic acid (Riberau-Gayon, Glories, Duboudieu, 2003). Tartaric acid and malic acid will occur in varying quantities dependent on varietal, vintage conditions and vineyard management (Jackson, 2008). Total acidity will be higher in cool rather than warm climates (Jackson, 2008). Malic acid as a proportion of total acidity will be higher in cool rather than warm climates (Jackson, 2008).

There are several methods for the deacidification of must and wine, Chemical (C) and Biological (B).
Table 1, Common Deacidification practices

Single Salt (C)

Double Salt (C)

Maloethanol Fermentation (B) Y N Y N

Malolactic Fermentation (B) Y N N Y

Removes Malic Acid Removes Tartaric Acid Pre-fermentation Post-fermentation

N Y Y Y

Y Y Y Y

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Single Salt Deacidification

Single salt deacidification is the removal of tartaric acid by neutralisation with a base substance producing a tartrate salt, carbon dioxide and water (Rankine, 2004).
Figure 1, Simple Equation of Single Salt Deacidification

Tartaric acid (C4H6O6)

Calcium carbonate (CaCO3)

Calcium tartrate (CaC4H4O6)

Carbon dioxide (CO2)

Water (H2O)

Single salt deacidification will not remove malic acid, only tartaric acid. As only a proportion of the acidity will be tartaric acid, to obtain an accurate deacidification the tartaric acid content rather than the total acidity as tartaric equivalent must be known (Gadek etal, 1980). A portion of the must or wine is deacidified to remove all of the tartaric acid content, after the salt has settled from solution the deacidified wine is returned to the bulk by racking, or preferably, filtration. This helps prevent over deacidification and the excessive removal of tartaric acid which would ruin the wine (Gadek etal, 1980).

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Double Salt Deacidification

Double salt deacidification is the removal of tartaric and malic acids by neutralisation with a base substance seeded with a tartarte-malate salt producing a tartrate-malate salt, carbon dioxide and water.
Figure 2, Simple Equation of Double Salt Deacidification

Tartaric acid (C4H6O6)

Malic acid (C4H6O5)

Calcium carbonate (CaCO3)

Calcium tartratemalate (Ca2C8H10O10)

Carbon dioxide (CO2)

Water (H2O)

Malic acid alone will not form a salt with a base. Double salt deacidification will remove both malic and tartaric acid but only if carried out correctly. Theoretically tartaric and malic acid should be removed in equal parts and malic acid can only be removed whilst tartaric acid is present to form the tartrate-malate salt. To obtain an accurate deacidification the tartaric acid content, rather than total acidity as tartaric equivalent must be known (Steele & Kunkee, 1978). The double salt deacidification requires the base / must solution to be kept above pH4.5 to allow the decarboxylation (removal of carbon) of malic acid and subsequent formation of the tartrate-malate salt (Steele & Kunkee, 1978). The deacidified must requires filtration as returning the tartrate-malate salt to the bulk must will cause the salt to dissolve into solution and the formation of a tartrate salt (Mattick,Plane, La Weirs, 1980).

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Maloethanol Fermentation
Maloethanol fermentation (MEF) is the process by which yeast utilise malic acid as a carbon source and subsequently produce ethanol. Malic acid is decarboxylated to pyruvate which is further decarboxylated to ethanol (Volschenk etal, 2001).
Figure 3, Simple Diagram of MEF in a yeast cell

Different yeast species have different capacities to take on and metabolise malic acid due to their production of differing malic acid enzymes (Pretorius, 2000). Most S. cerevisiae are poor malic acid metabolisers and carry out no MEF Volschenk etal, 2001), non-sacc. yeasts Schizosaccharomyces pombe & Issatchenkia orientalis are both able to consume malic acid up to 30g/l (Taillandier & Strehaiano, 1991; Seo, Rhee, Park, 2007).

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Malolactic Fermentation
Malolactic fermentation (MLF) is the process by which bacteria utilise malic acid as a carbon source producing lactic acid by decarboxylation (Versari, Parpinello, Cattaneo, 1999). Oenococcus oeni is the most prevalent commercial MLF bacteria due to a comparatively high tolerance to pH, acidity, malic acid, ethanol, temperature and total SO2, though these tolerances are all considerably lower than yeasts used for alcoholic fermentation (AF) (Sauvageot & Vivier, 1997).
Table 2, Common optimum parameters for MLF (Versari, Parpinello, Cattaneo, 1999).

Total SO2 <30ppm

Temperature 18-20C

Alcohol <11%

pH >3

Malic acid <4g/l

As MLF requires more hospitable conditions than may be common at the end of AF in the UK additional or prior chemical deacidification may still be required. Diacetyl (buttery, creamy aroma) is a common secondary product of MLF and may not be desirable in a light aromatic wine. MLF inoculum that produce low or no diacetyl such as CiNe (www.chrhansen.com, 2011) or Malostar Fruit (www.lalittorale.de) have shown poor results outside of optimal conditions.

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S. cerevisiae
S. cerevisiae is the most common wine making yeast as it is tolerant to high sugar, acid and ethanol environments, ferments to desired alcohol levels and produces low levels of fault (Rankine, 2004). Though S. cerevisiae is able to consume malic acid (Radler & Fuck, 1970) it is considered to be a poor consumer (Volschenk etal, 2001; Volschenk etal, 1997) Malic acid enters the cell via passive diffusion The malic acid enzyme is located within the mitochondria providing energy exclusively for the mitochondria The malic acid enzyme has a low affinity for the substrate S. cerevisiae with improved abilities to convert malic acid to ethanol such as Lalvin 71B and Lalvin ICV-GRE (33% and 18% conversion respectively) have been isolated and are commercially available (Main, Threlfall, Morris, 2007)

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Sz. pombe
Sz. Pombe yeast perform both alcoholic fermentation (AF) of sugars and the decarboxylation of malic acid to ethanol (Portugal etal, 2011). Fermentation with Sz. pombe alone has been demonstrated to produce unpleasant aroma (Snow & Gallander, 1979; Gallander, 1977). Encapsulation of yeast using alginate beads allows the transfer of nutrient, sugars and acid into the yeast and the excretion of ethanol whilst preventing the yeast cells from multiplying or becoming free within the ferment (Portugal etal, 2011). The use of encapsulated yeast allows for easy removal of the yeast mass from the ferment after the required MEF has occurred without the production of organoleptic fault.

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Sequential Inoculation
Sequential inoculation with encapsulated Sz. pombe followed by S. cerevisiae has been demonstrated to provide reliable malic reduction, reliable alcoholic fermentation and no organoleptic fault (Portugal etal, 2011; Yokotsuka, 1993).

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Trials
After cold settling and enrichment measure pH, Total acidity (TA), Tartaric Acid (TrA), Malic acid (MA), and specific gravity (SG). Wines should be monitored throughout ferment. Control (No Deacidification) Double Salt Deacidification Malolactic Fermentation Maloethanol Fermentation

1 month post bottling wines should be chemically and organoleptically assessed. Analysed in triplicate for alcohol by volume, residual sugar, pH, total acidity, tartaric acid, malic acid, lactic acid and volatile acidity. Organoleptically, wines should be tasted blind by a panel for preference comparing all wines for all panellists 6 tests each in total, followed by quantitative descriptive analysis for each wine (Iland etal, 2004).

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Trials - Control
Label 3 demi-johns as C1, C2, C3. Fill demi-johns (sterilised with very hot water) with 4L each of must. Inoculate with preferred yeast to manufacturer s instruction. Fit airlocks and ferment in a convenient location (approx. 15C). Monitor organoleptics and SG daily, sterilise all equipment (i.e. hydrometer, thermometer, syphon) before and after each use to prevent cross contamination of ferments When wine is below 1.05SG move to a warmer environment (approx. 20C) to complete fermentation. When wine is below 0.99SG add 50ppm SO2 After settling rack off lees. Make protein and tartrate stable. Adjust SO2 and bottle and label for future testing.

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Trials Double Salt

Label 3 demi-johns as DS1, DS2, DS3. Deacidify 20L of must with Acidex as per manufacturer s instruction, if available a table top pad filter would ensure the tartrate-malate salt is removed from the deacidified portion. Fill demi-johns (sterilised with very hot water) with 4L each of must. Inoculate with preferred yeast to manufacturer s instruction. Fit airlocks and ferment in a convenient location (approx. 15C). Monitor organoleptics and SG daily, sterilise all equipment (i.e. hydrometer, thermometer, syphon) before and after each use to prevent cross contamination of ferments When wine is below 1.05SG move to a warmer environment (approx. 20C) to complete fermentation. When wine is below 0.99SG add 50ppm SO2 After settling rack off lees. Make protein and tartrate stable. Adjust SO2 and bottle and label for future testing.

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Trials - MLF
Label 3 demi-johns as MLF1, MLF2, MLF3. Fill demi-johns (sterilised with very hot water) with 4L each of must. Inoculate with preferred yeast to manufacturer s instruction. Fit airlocks and ferment in a convenient location (approx. 15C). Monitor organoleptics and SG daily, sterilise all equipment (i.e. hydrometer, thermometer, syphon) before and after each use to prevent cross contamination of ferments When wine is below 1.05SG move to a warmer environment (approx. 20C) to complete fermentation. When wine is below 0.99SG begin MLF with your preferred culture as per manufacturer s instruction. Post MLF add SO2 at 50ppm. After settling rack off lees. Make protein and tartrate stable. Adjust SO2 and bottle and label for future testing.

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Trials - MEF
Label 3 demi-johns as MEF1, MEF 2, MEF 3. Fill demi-johns (sterilised with very hot water) with 4L each of must. Inoculate with ProMalic as per manufacturer s instruction. Fit airlocks and ferment in a convenient location (approx. 15C). Monitor organoleptics, SG, pH and TA daily, sterilise all equipment (i.e. hydrometer, thermometer, syphon) before and after each use to prevent cross contamination of ferments. SG should remain constant, acidity should reduce by organoleptic and chemical analysis. Day 5 inoculate with preferred yeast to manufacturer s instruction. Day 10 (or earlier if fault is noted) remove ProMalic beads. When wine is below 1.05SG move to a warmer environment (approx. 20C) to complete fermentation. When wine is below 0.99SG add 50ppm SO2 After settling rack off lees. Make protein and tartrate stable. Adjust SO2 and bottle and label for future testing.

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Monitoring
Table 3, Example of Trial Monitoring Sheet

Ferment

Day

SG

pH

TA

Organoleptic

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The Future
Advances in gene manipulation have allowed the development of genetically modified yeast for wine production. ML01; S. cerevisiae + Sz. pombe + O. oeni

ML01 is an S. cerevisiae yeast with a Sz. pombe malic permease gene (mae1) and O. oeni malolactic gene (mleA), this allows the yeast to carry out both AF and MLF. It is approved for use in the USA and several other countries under GRAS (Generally Regarded As Safe) but is currently illegal to use in the EU (Husnick etal, 2007) ME01? S. cerevisiae + Sz. Pombe / I.orientalis

Research continues on the development of malic degrading yeast with lab trials being carried out on S. cerevisiae with Sz. Pombe and I. orientalis genes allowing the active transport of malic into the yeast cell and subsequent conversion to alcohol (Kim, Hong, Park, 2008; Volschenk etal, 2001). Demalication by nanofiltration, removal 34% (average) of malic acid via tangential filtration (Ducruet, Fast-Merlier, Noilet, 2010)

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References
Amerine, MA; Roessler, EB; Ough, CS; (1965), Acids and the Acid Taste. I. The Effect of pH and Titratable Acidity, American Journal of Enology & Viticulture, v16i1 Chr-hansen; (2011), Viniflora CiNe Inoculation & Usage Protocol, last accessed 27/12/2011 at http://www.chrhansen.com/uploads/tx_tcdownloadables/Viniflora_CiNe_inoculation_protocol_and_guidelines_Oct_2010.pdf Drysdale, GS; Fleet, GH; (1988), Acetic Acid Bacteria in Winemaking: A Review, American Journal of Enology & Viticulture, v39i2 Ducruet, J; Fast-Merlier, K; Noilet, P; (2010), New Application for Nanofiltration:Reduction of Malic Acid in Grape Must, American Journal of Enology & Viticulture v61i2 Edwards, CG; Reynolds, AG; Rodriguez, AV; Semon, MJ; Mills, JM; (1999), Implication of Acetic Acid in the Induction of Slow/Stuck Grape Juice Fermentations and Inhibition of Yeast by Lactobacillus sp, American Journal of Enology & Viticulture, v50i2 Gadek, HJ; Diamond, F; Mearnet, M; McMullin, M; Szvetecz, MA; Verano, FP; (1980), Preliminary Investigation of Deacidification Methods and Carbonic Maceration of French Hybrid Wines, American Journal of Enology & Viticulture, v31i1 Gallander, JF, (1977), Deacidification of Eastern Table Wines with Schizosaccharomyces pombe, American Journal of Enology & Viticulture, v28 i2 Henderson, P; (2009), Sulphur Dioxide; Sxience behind this anti-microbial, anti-oxidant, wine additive, Practical Winery & Vineyard Journal Husnik, JI; Delaquis, PJ; Cliff, MA; van Vuuren, HJJ; (2007), Functional Analyses of the Malolactic Wine Yeast ML01, American Journal of Enology & Viticulture, v58i1 Iland, P. Bruer, N. Edwards, G. Weeks, S. & Wilkes, E. (2004). Chemical Analysis of Grapes and Wine: Techniques and Concepts, Campbelltown SA Australia, Patrick Iland Wine Promotions Pty Ltd Jackson, RS; (2008), 3rd Ed, Wine Science, Principles and Applications, London, Elsevier Kim, DH; Hong, YA; Park, HD; (2008), Co-fermentation of grape must by Issatchenkia orientalis and Saccharomyces cerevisiae reduces the malic acid content in wine, Biotechnology Letters, v30 Lalittorale; (2011), Malostar Fruit, last accessed 27/12/2011 at http://www.lalittorale.de/en/Datenblatt/MaloStar_Fruit.pdf Lallemand wine; (2011), Evaluating MLF viability, last accessed 27/12/2011 at http://www.yapak.fr/lallemandoeno/ Main, GL; Threlfall, RT; Morris, JR; (2007), Reduction of Malic Acid in Wine Using Natural and Genetically Enhanced Microorganisms, American Journal of Enology & Viticulture, v58i3 Portugal, I; Ribeiro, SC; Xavier, AMRB; Centeno, F; Strehaiano, P; (2011), Immobilised yeast grape must deacidification in a recycle fixed bed reactor, International Journal of Food Science & Technology, v46 Mattick, LR; Plane, RA; La Weirs, VD; (1980), Lowering Wine Acidity with Carbonates, American Journal of Enology & Viticulture, v31i4 Pretorius, IS; (2000), Tailoring wine yeast for the new millennium: novel approaches to the ancient art of winemaking, Yeast, v16 Riberau-Gayon, P; Glories, Y; Duboudieu, D; (2003) Handbook of Enology Volume 2; The chemistry of wine stabilization and treatments, West Sussex, John Wiley & Sons Ltd Radler, F; Fuck, E; (1970), Conversion of L-malic acid during Saccharomyces cerevisiae fermentation, Experientia 26:731 Rankine, B; (2004), Making Good Wine, Macmillan, Australia Sauvageot, F; Vivier, P; (1997), Effects of Malolactic Fermentation on Sensory Properties of Four Burgundy Wines, American Journal of Enology & Viticulture, v48i2 Scott Laboratories; (2011), Promalic technical data sheet, last accessed 27/12/2011 at http://www.scottlab.com/uploads/documents/downloads/269/ProMalic%206-16-10.pdf Seo, SH; Rhee, CH; Park, HD; (2007), Degradation of Malic Acid by Issatchenkia orientalis KMBL 5774, an Acidophilic Yeast Strain Isolated from Korean Grape Wine Pomace, The Journal of Microbiology, v45i6 Snow, PG; Gallander, JF; (1979), Deacidification of White Table Wines Through Partial Fermentation with Schizosaccharomyces pombe. American Journal of Enology & Viticulture, v30 Steele, JT; Kunkee, RE; (1978), Deacidification of Musts from the Western United States by the Calcium Double-Salt Precipitation Process, American Journal of Enology & Viticulture, v29i3 Taillandier, P; Strehaiano, P; (1991), The role of malic acid in the metabolism of Schizosaccharomyces pombe: substrate consumption and cell growth, Applied Microbiology & Biotechnology v35 Versari, A; Parpinello, GP; Cattaneo, M; (1999), Leuconostoc oenos and malolactic fermentation in wine: A review, Journal of Industrial Microbiology & Biotechnology v23 Volschenk, H; ViljoenBloom, M; Subden, RE; VanVuuren, HJJ; (2001), Malo-ethanolic fermentation in grape must by recombinant strains of Saccharomyces cerevisiae, Yeast, v18 Yokotsuka, K; Otaki, A; Naitoh, A; Tanaka, H; (1993), Controlled Simultaneous Deacidification and Alcohol Fermentation of a High-Acid Grape Musts Using Two Immobilized Yeasts, Schizosaccharomyces pombe and Saccharomyces cerevisiae, American Journal of Enology & Viticulture, v44 i4

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