New Aspects in Wine Technology and Process Engineering

New Aspects in Wine Technology and Process Engineering Prof.Dr.Marin Berovic Faculty of Chemistry and Chemical Engineering Department of Chemical and Biochemical Engineering

Stirred Tank Reactor and instrumentation for on-line process control

Metabolism of Saccharomyces cerevisiae

ALCOHOL FERMENTATION

Metabolism of Saccharomyces cerevisiae media cytoplasma glucose glucose-6-fosfate glicolysis glicerolaldehyd-3-fosfate dihydroxiaceton fosfate NAD + aldolase NADH NADH 3-isophosphate isomerase NAD + 1,3-difosfoglicerate glicerol-3-fosfate NADH NAD + piruvate acetaldehide ethanol glicerol

Methods and possibilities for on-line control of metabolism of Saccharomyces cerevisiae

Redox Potential in Wine Technology BEROVIC, Marin, MAVRI, Jan, WONDRA, Mojmir, KOSMERL, Tatjana, BAVČAR, Dejan. Influence of temperature and carbon dioxide on fermentation of Cabernet Sauvignon must. Food technol. biotechnol., 3, vol. 41, no. 4, 353-359 PIVEC, Aleksandra, BEROVIC, Marin, CELAN, Stefan, WONDRA, Mojmir. The role of on-line redox potential measurement in Sauvignon blanc fermentation. Food technol. biotechnol.,, vol. 4, no. 1, 49-55. PIVEC, Aleksandra, BEROVIC, Marin, WONDRA, Mojmir, CELAN, Štefan, KOŠMERL, Tatjana. Influence of temperature shock on the glycerol production in cv. Sauvignon blanc fermentation. Vitis, 3, vol. 4, no. 4, 5-6

REDOX POTENTIAL E = E calomel + RT/ nf ln a oxidants / a reductants E calomel = 44 mv E Standard redox potencial E h = E + RT/ nf ln a oxidants / a reductants Clark rh potential definition rh =.343 Eh + ph ; rh = - log ph ; ph = - log ah

Redox potential in more complex systems k k j j i i S S ze S S 1 1 ox red ox i red i o h a a zf RT E E ln i i i S S S i i a a a a 1 1 Product of activities of all the oxidants or reductants in the system Π a νί oxidants / reductants

Redox electrode Pt / calomel ph electrode glass/calomel

REDOX POTENTIAL in WINE Wine represent complex oxydo-reductive system composed by : Chemical oxydo reducitive compounds : alcohols, aldehydes, organic (oxo & keto) acids polyphenols, kinons, ascorbic & dehydroascorbic acid, Ion couples Fe + /Fe 3+ ; Cu + /Cu + Redox potential in wine express intensivity and the rate of the sum of all oxydo - reductive processes Metabolic activity of microbial culture and its metabolism play the main role sugars (glucose) alcohols + biomass

REDOX CHARACTERISTICS of WINES Premium quality wines are characterized by the lowest redox potential values Reductive and close character wines are disposed to oxidation Reductive non-oxydized wines Eh = 1-18 mv ; rh=18 do Oxydized wines Eh = 68-3 mv ; rh=4 do 5 White bottled wines : Eh = 75-8 mv Red bottled wines : Eh = 1-3 mv REDUCTANTS SO, ascorbic acid, cell products (cisteine, glutatione) are parameters that are decreasing redox potential and express reductive character OXIDANTS Air oxygen, bentonites, mineral adsorbents, peroxyde PROTECTORS AND ANTIOXIDANTS IN FERMENTATION Carbone dioxyde, gaseous nitrogen are protective agents in the pipes and fermentor head space

Sulphurization Lactic fermentation 1 Flow Optimal colour Altered Oxydized Sheryzation Mousyness Acetic flavour Redox potential and sensorial analysis in wine fermentation Eh mv Aerobic fermentation 3 Brownish Oxidized taste Flow Intensive yellow Optimal taste 1 Stabilization Filtration Sulphurization Bottling Light yellow Non intensive taste Young wine Wine in the barrell Bottled wine Month Unsulphurized grape must Sulphurized grape must Reductive non-oxydized wines Eh = 1-18 mv ; rh=18 do Oxydized wines Eh = 68-3 mv ; rh=4 do 5 White bottled wines : Eh = 75-8 mv Red bottled wines : Eh = 1-3 mv

15 1 5 Eh (mv) Redox potential of unsulfurized Sauvignon blanc must alcohol fermentation Aerobic phase Anaerobic phase Wine maturation - 1 mv Eh / (mv) -5 4 48 7 96 1 144 168 19 16 4 64 88 31 336 36-1 -15 - alcohol fermentation 15-15 mv Fermentation time (h) čas fermentacije / (h) Fermentation temperature 18 o C

Temperature influence T [ C] E h [mv] - starting E h [mv] - final ΔE h [mv] 15 1-1 18-15 35 4-4 5 Eh (15) Eh (18) Eh (4) 15-5 Eh / (mv) 1 5-5 4 48 7 96 1 144 168 19 16 Eh / mv -1-15 -1 - -15 - -5 15 16 17 18 19 1 3 4 5-5 čas Fermentation fermentacije (h) / (h) T / o C Redox potential at 15, 18 in 4 C Eh [mv] - final as a function of temperature

1 15 ºC 18 ºC Eh / mv; red. sladkor, etanoll / gl - 35 Eh sladkor etanol acetaldehid,45,4 5,35 15,3,5 5, -5,15,1-15,5-5 1 3 4 5 6 7 8 9 1 11 1 čas Fermentation fermentacije (day) / dan acetaldehid / gl -1 Eh / mv, red. sladkor, etanol / gl -1 5 15 1 5-5 -1-15 - -5 Eh sladkor etanol acetaldehid 1 3 4 5 6 7 8 9 1 11 1 čas fermentacije / dan Fermentation (day),45,4,35,3,5,,15,1,5 acetaldehid / gl -1 4 ºC Eh / mv; red. sladkor, etanol / gl -1 5 Eh sladkor etanol acetaldehid,45,4 15,35 1,3 5,5, -5-1,15-15,1 -,5-5 1 3 4 5 6 7 8 9 1 11 1 acetaldehid / gl -1 Fermentation (day) čas fermentacije / dan Redox potential, reducitive sugars consumtion, acetaldehyde and ethanol at 15, 18 in 4 C

glicerol / (gl -1 ) Temperature influence on redox potential and glycerol biosynthesis 5 15 Eh (15) Eh (18) Eh (4) glicerol(15) glicerol(18) glicerol(4) 9 8 7 Eh / (mv) 1 5-5 -1-15 - -5 6 5 4 48 7 96 1 144 168 19 16 4 644 3 1 čas Fermentation fermentacije /(day) (h) Fermentation temperatures 15, 18 and 4 C

glicerol / (gl -1 ) glicerol / (gl -1 ) Redox potential and glycerol in sulfurized grape must Eh / (mv) 5 Eh(4)-S Eh(18)-S Eh(15)-S glicerol(4)-s glicerol(18)-s glicerol(15)-s 9 8 15 7 1 6 5 5 4 48 7 96 1 144 168 19 16 44-5 3-1 -15 - Redoks potencial in nastanek glicerola pri fermentacijah -5 z dodano žveplovo(iv) kislino pri 15, 18 in 4 ºC čas Fermentation fermentacije / (h) (h) 1 9 8 7 6 5 4 3 1 glicerol(15)-s glicerol(18)-s glicerol(4)-s glicerol(15) glicerol(18) glicerol(4) 4 48 7 96 1 144 168 19 16 4 64 Fermentation čas fermentacije / (h) (h) Fermentation temperatures pri 15, 18, 4 C

5 15 1 E (15)-S glukoza fruktoza etanol acetaldehid,7,6,5 acetaldehid / gl -1 Consumption of glucose and fructose, production of ethanol in sulfurized grape must Eh / mv 5-5,4,3 15 C -1, -15,1 - -5 1 3 4 5 6 7 8 9 1 11 čas Fermentation fermentacije / (h) dan 5 15 1 E (18)-S glukoza fruktoza etanol acetaldehid,7,6,5 18 C. Eh / (mv) 5-5,4,3 acetaldehid / gl -1-1, 5 Eh(4)-S glukoza fruktoza etanol acetaldehid,7-15 -,1 15 1,6,5 acetaldehid / gl -1-5 1 3 4 5 6 7 8 9 1 11 čas Fermentation fermentacije (h) / dan Eh / mv 5-5 -1,4,3, 4 C. -15 -,1-5 1 3 4 5 6 7 8 9 1 11 čas Fermentation fermentacije / (h) dan

Redox potential in other cultivar fermentation

Eh (mv) Eh(mV) Redox potential in Cabernet sauvignon fermentation 15 1 f-a f-b 5-5 7 14 1 8 35 4 49 56 63 7 77-1 -15 - čas(h) Fermentation with and without additional CO 15 1 5-5 -1-15 - -5-3 1 3 4 5 6 7 8 čas (h) f-18 f- f-6 f- apr Fermentation with additional CO at T 18,, 6

E / (mv); sugar, EtOH / (g/l) biomass / (g/l) E / (mv); sugar, EtOH / (g/l) biomass / (g/l) E / mv ; red.sugars/ (g/l), Ethanol / (g/l) biomass / (g/l) Sauvignon blanc fermentation 3 7 5 6 5 15 1 4 3 5-5 4 48 7 96 1 144 168 19 16 4 64 88 1 fermentation time / h T 15 C 3 5 8 7 6 15 1 5 4 5-5 -1 4 48 7 96 1 144 168 19 16 4 64 88 3 1 fermentation time / (h) T 18 C 3 1 8 7 6 5-1 - -3 4 48 7 96 1 144 168 19 16 4 64 88 4 3 1 fermentation time / (h) T 4 C

Blue frankish fermentation T 4 C on-line data With CO injection Fermentation (h)

Influencing factors for regulation of Saccharomyces cerevisiae metabolism

The Influence of Galvanic Field on Saccharomyces cerevisiae in Grape Must Fermentation M. BEROVIC 1), M. POTOCNIK 1) and J. STRUS ) Vitis 47 (), 117-1 (8)

GALVANIC FIELD

S. cerevisiae in fermentation with electrostimulation Current 3 µa, after 7 hours, colourized with methylen blue

Eh (mv) Eh (mv) The influence of galvanic field on Saccharomyces cerevisiae in grape must fermentation 45 6 45 3 3 15 15-15 4 6 8 1 1 4 6 8 1 1-15 -3-45 -3 Fermentation time ( days ) -6 Fermentation time ( days ) Fermentation on-line redox potential courses T = 18, and 6 C, without electrostimulation T = 6 C ( 1,3 µa), T = 6 C ( 7,7 µa), T = 6 C ( 3 µa), with electrostimulation.

Glucose (g/l) Biomass (g/l) Fructose (g/l) The influence of galvanic field on Saccharomyces cerevisiae in grape must fermentation 1 14 8 6 1 1 6 4 4 8 6 4 4 6 8 1 Fermentation time (days) 1 3 4 5 6 7 8 4 6 8 1 Fermentation (days) Fermentation (days)

koncentracija (g/l) Acetaldehyde (g/l) koncentracija Concentration (g/l) (g/l) Concentration (g/l) koncentracija Concentration Concentration (g/l) koncentracija (g/l) koncentracija Concentration (g/l) (g/l) Lactic acid Nastajanje mlečne kisline med fermentacijo (g/l) F -temp. 18 C F -temp. C F - temp. 6 C F- temp. 6 C - ES (1,3 µa;,5 V/cm) F- temp. 6 C - ES (7,7 µa;,9 V/cm) F- temp. 6 C - ES (3 µa; 1,7 V/cm) 4,5 4 3,5 3,5 1,5 1,5 4 6 8 1 1 Fermentation čas (dni) time (days) Upadanje koncentracije jabolčne Malic kisline acid med fermentacijo (g/l) F -temp. 18 C F -temp. C F - temp. 6 C F- temp. 6 C - ES (1,3 µa;,5 V/cm) F- temp. 6 C - ES (7,7 µa;,9 V/cm) F- temp. 6 C - ES (3 µa; 1,7 V/cm) 1 8 6 4 4 6 8 1 1 Fermentation time (days) čas (dni) Nastajanje etanola Ethanol med fermentacijo (g/l) F -temp. 18 C F -temp. C F - temp. 6 C F- temp. 6 C - ES (1,3 µa;,5 V/cm) F- temp. 6 C - ES (7,7 µa;,9 V/cm) F- temp. 6 C - ES (3 µa; 1,7 V/cm) 1, 8, 6, 4,,, 4 6 8 1 1 čas (dni) Fermentation time (days) 1 1 8 6 4,4,3,,1 4 6 8 1 1 Fermentation čas (dni) time (days),5,4,3,,1 F -temp. 18 C F -temp. C F - temp. 6 C F- temp. 6 C - ES (1,3 µa;,5 V/cm) F- temp. 6 C - ES (7,7 µa;,9 V/cm) F- temp. 6 C - ES (3 µa; 1,7 V/cm) F -temp. 18 C F - temp. 6 C Glycerol Iso-amilalcohol,, -,1, 4, 6, 8, 1, 1, čas (dni) F -temp. C F- temp. 6 C - ES (1,3 µa;,5 V/cm) F- temp. 6 C - ES (7,7 µa;,9 V/cm) F- temp. 6 C - ES (3 µa; 1,7 V/cm) Fermentation time (days) Acetaldehyde,,, 4, 6, 8, 1, Fermentation time (days)

MAGNETISM & MAGNETIC FIELD

Number cells / ml Number cells / ml Number cells/ ml Influence of static magnetic field on Saccharomyces cerevisiae metabolism PRIMERJAVA ŠTEVILA CELIC 1,E+7 9,E+7 8,E+6 6,E+6 4,E+6 control kontrolna magnetic kultura field magnetno polje 7,E+7 5,E+7 3,E+7 control kontrolna kultura magnetic field magnetno polje,e+6 1,E+7,E+ -1,E+7 1 day days PRIMERJAVA ŠTEVILA CELIC 1 9,E+8 8 1,6E+8 1,E+8 8,E+7 control kontrolna kultura magnetic field magnetno polje 7 6 5 4 3 1 dan dni 3 dni 4,E+7,E+ 3 days 1 1 dan 3 Exp.days

Concentration (g/l) Concentration (g/l) Concentration (g/l) Concentration (g/l) Influence of static magnetic field on Saccharomyces cerevisiae metabolism ETHANOL ISOAMIL ALCOHOL 1 9 8 7 6 5 4 3 1 5 1 15 5 3 Fermentation (h) kontrolna control kultura mag. 1.day ploje 1 dan mag..day polje dni mag. 3.day polje 3 dni,45,4,35,3,5,,15,1,5 -,5 5 1 15 5 3 Fermentation (h) kontrolna control kultura mag. 1.day polje 1 dan mag..day polje dni mag. 3.day polje 3 dni 1- PROPANOL - BUTANOL,5,,15,1,5 5 1 15 5 3 -,5 Fermentation (h) kontrolna control kultura mag. 1.day polje 1 dan mag..day polje dni 3.day mag. polje 3 dni,35,3,5,,15,1,5 -,5 5 1 15 5 3 Fermentation (h) kontrolna control kultura mag.polje 1.day 1 dan mag. polje.day dni 3.day mag. polje 3 dni

Fermentation (h) Concentration (g/l) Influence of static magnetic field on Saccharomyces cerevisiae metabolism BIOMASS ACETALDEHYDE 5 4 3 1 5 1 15 Concentration g/l kontrolna control kultura mag. polje 1.day 1 dan mag. polje.day dni mag. polje 3.day 3 dni,4,35,3,5,,15,1,5 -,5 5 1 15 5 3 Fermentation (h) control kontrola kultura 1.day mag. polje 1 dan mag. polje.day dni mag. polje 3.day 3 dni

Concentration (g/l) Concentration (g/l) Concentration (g/l) Concentration (g/l) Influence of static magnetic field on Saccharomyces cerevisiae metabolism GLUCOSE FRUCTOSE 9 8 7 6 5 4 3 1 5 1 15 5 3 Fermentation (h) kontrolna control kultura mag. polje 1.day 1 dan.day mag. polje dni 3.day mag. polje 3 dni 14 1 1 8 6 4 5 1 15 5 3 Fermentation (h) kontrolna control kultura mag. 1.day polje 1 dan mag..day polje dni mag. 3.day polje 3 dni TARTARIC ACID MALIC ACID 8 7 6 5 4 3 1 5 1 15 5 3 Fermentation (h) kontrolna control kultura mag. 1.day polje 1 dan mag..day polje dni mag. 3.day polje 3 dni 7 6 5 4 3 1 5 1 15 5 3 Fermentation (h) kontrolna control kultura mag. 1.day polje 1 dan mag..day polje dni mag. 3.day polje 3 dni

Concentration (g/l ) Concentration (g/l) Influence of static magnetic field on Saccharomyces cerevisiae metabolism LACTIC ACID GLYCEROL 4 3,5 3,5 1,5 1,5 5 1 15 5 3 control kontrolna kultura 1.day mag. polje 1 dan.day mag. polje dni 3.day mag. polje 3 dni 1 1 8 6 4 5 1 15 5 3 control kontrolna kultura mag. 1.day polje 1 dan mag..day polje dni mag. polje 3 dni 3.day Fermentation (h) Fermentation (h)

Glycolisis

FERMENTATION TEMPERATURE

Berovic M, Herga M. 7 Heat shock on Saccharomyces cerevisae inocolum increases glycerol production in wine fermentation Biotechnol. Lett. vol. 9, 6, 891-894 (7) Berovic M, Pivec A., Kosmerl T., Wondra M., Celan S., Heat Shock on Glycerol Production in Alcohol Fermentation. J.Biosc.Bioeng.vol.13,,135-139 (7)

Single heat shock 35 18 C 34 C 18 C 3 5 6 h 4 h 1 h Eh / (mv) 15 1 5 Redox potential and tempetature profile at single heat shock up to 34 C 4 48 7 96 1 144 168 19 16-5 -1-15 Fermentation (h) 5 sladkor(tš) etanol(tš) E(TŠ) acetaldehid(tš),5,45 Redox potential, reductive sugars, acetaldehyde, ethanol, at single heat shock up to 34 C Eh / mv; sladkor, etanol / gl -1 15 1 5,4,35,3,5, acetaldehid / gl -1-5 -1,15,1,5-15 1 3 4 5 6 7 8 9 1 11 1 Fermentation čas fermentacije (day) / dan

Double heat shock 34 C 34 C 35 18 C 18 C 5 Eh / mv 15 5 Redox potential and tempetature profile at double heat shock up to 34 C 4 48 7 96 1 144 168 19 16 4-5 -15 čas Fermentation fermentacije / (min) 5 etanol(dtš) sladkor(dtš) E(DTŠ) acetaldehid(dtš),5 Eh / mv; sladkor, etanol / gl -1,45 Redox potential, reductive sugars, acetaldehyde, ethanol, at double heat shock up to 34 C 15 1 5-5 -1,4,35,3,5,,15,1,5 acetaldehid / gl -1-15 1 3 4 5 6 7 8 9 1 11 1 Fermentation čas fermentacije (day) / dan

On-line redox potential at single, double heat shock and in control in Sauvignon blanc fermentation 5 15 1 ESingle h -TŠ heat shock EControl h -BTŠ EDouble h -DTŠ Eh / (mv) 5-5 144 88 43 576 7 864 18 115 196 144-1 -15 - čas fermentacije Fermentation(h) / (min)

Influence of heat shock on glycerol production 5 1 Glycerol (g/l) E(TŠ) E(DTŠ) E(BTŠ) glicerol(tš) glicerol(dtš) glicerol(btš) 15 1 1 8 glicerol / gl -1 Eh / mv 5-5 -1 6 4-15 1 3 4 5 6 7 8 9 Fermentation čas fermentacije (day) / dan

Glicerol Glycerol (g/l) Glycerol (g/l) Glycerol (g/l) Glycerol (g/l) Influence of single and double heath shock on glicerol production 1 1 1 8 8 6 6 4 4 5 1 15 5 5 1 15 Fermentation time (h) (h) Fermentation time (h) (h) Fig. 1: Glicerol production at constant fermentation temperature of 18ºC; black square, control; black circle, single heat shock at 34ºC for 4 h; black triangle, single heat shock at 34ºC for 4 h. Fig. : Glicerol production at constant fermentation temperature of 18 ºC; black square, control; black circle, double heat shock at 34ºC ( x 4 h); and black triangle, double heat shock at 34ºC ( x 8 h).

1-propanol / (gl -1 ) izoamilalkohol / (gl -1 ) jantarna kislina / (gl -1 ) ocetna kislina / (gl -1 ),9,8,8,7,7,6,6,5,5,4,4,3,3,,,1 jantarna(btš) jantarna(tš) jantarna(dtš),1 4 48 7 96 1 144 168 19 16 4 Fermentation (h) čas fermentacije / (h) Succinic acid ocetna(btš) ocetna(tš) ocetna(dtš) 4 48 7 96 1 144 168 19 16 4 Fermentation (h) čas fermentacije / (h) Acetic acid,45,4,35 1-propanol(BTŠ) 1-propanol(TŠ) 1-propanol(DTŠ),6,5 izoamilalkohol(btš) izoamilalkohol(tš) izoamilalkohol(dtš),3,4,5,,15,3,,1,5 4 48 7 96 1 144 168 19 16 4 čas fermentacije / Fermentation (h) 1-propanol,1 4 48 7 96 1 144 168 19 16 4 čas fermentacije / (h) Fermentation (h) Iso-amil alcohol

Metabolites produced in fermentations at various temperature conditions Single HS: single heat shock for 4 h and single heat shock for 4 h Double HS: double heat shock for x 4 h and double heat shock for x 8 h. Fermentation Reduc. sugars (g l -1 ) Isoamyl alcohol (g l -1 ) 1- propanol (g l -1 ) Acet aldehyde (g l -1 ) Ethanol g l -1 ) E ( Glycerl (g l -1 ) Acetic acid (g l -1 ) Succ. acid (g l -1 ) Citric acid (g l -1 ) Malic acid (g l -1 ) Tartaric acid (g l -1 ) Control (18 C) 3..4.9.9 86.9 6.7.35.67.58.9.5 Single HS (4h).6.33.31.1 9. 7.9.45.73.57.5 3.5 Single HS (4h).3.35.33.1 94. 8..41.74.58.5 3.5 Double HS (x4h)..51.37.13 11. 1..56.78.7.3 3.6 Double HS (x8h) 1.9.53.39.14 13.11 1.9.56.79.7.1 3.5

glicerol (g/l) glicerol (g/l) Biosynthesis of glycerol at temperature oscilation Fermentation with 8 hour temperature oscilation (18 - C ) Fermentation with 8 h temperature oscilation (18-5 C ) 1 8 6 4 1 8 6 4 5 1 15 5 5 1 15 5 Fermentation (h) Fermentation (h)

Glicerol (gl -1 ) Biomass (g l -1 ) Glycerol (gl -1 ) Biomass (g l -1 ) Glycerol (gl -1 ) Influence of heat shock on Sacharomyces cerevisiae inoculum 8 1 8 6 1 1 1 6 1 4 8 4 5 1 15 5 Fermentation (h) 1 8 6 8 6 4 5 1 15 5 Fermentation (h) 6 4 4 5 1 15 5 Fermentation (h) 5 1 15 5 Fermentacja (h) 5 1 15 5 Fermentation (h) Fig.1. Production of biomass with inoculum exposed for min to the heat shock at 45 C and control at 18 ºC. Fig. Glycerol biosynthesis with inoculum exposed for min to the heat shock at 45 C and control at 18 ºC..

biomasa (g/l) etanol (g/l) Temp. šok Temp. šok 8 7 6 18 C temp. šok 45 C x temp. šok 34 C 1 1 18 C temp. šok 45 C x temp. šok 34 C 5 8 4 3 1 6 4 5 1 15 5 čas (h) 5 1 15 5 čas (h) Naraščanje biomase in etanola med fermentacijo pri 18ºC, minutnem temperaturnem šoku na vcepku pri 45ºC in dvakratnem 1 urnem temperaturnem šoku pri 34 ºC

glukoza (g/l) fruktoza (g/l) Temp. šok Temp. šok 1 1 8 18 C temp. šok 45 C x temp. šok 34 C 1 1 8 18 C temp. šok 45 C x temp. šok 34 C 6 6 4 4 5 1 15 5 - čas (h) 5 1 15 5 - čas (h) Poraba glukoze in fruktoze med fermentacijo pri 18ºC, minutnem temperaturnem šoku na vcepku pri 45ºC in dvakratnem 1 urnem temperaturnem šoku pri 34 ºC

izoamil alkohol (g/l) acetaldehid (g/l) Nastajanje izoamilalkohola, 1-propanola in acetaldehida med fermentacijo pri 18ºC, minutnem temperaturnem šoku na vcepku pri 45ºC in dvakratnem 1 urnem temperaturnem šoku pri 34 ºC 1-propanol (g/l) Temp. šok Temp. šok,7,6,5 18 C temp. šok 45 C x temp. šok 34 C,8,7,6 18 C temp. šok 45 C x temp. šok 34 C,4,3,,1 5 1 15 5 -,1 čas (h),5,4,3,,1 5 1 15 5 -,1 čas (h) Temp. šok,4,35,3 18 C temp. šok 45 C x temp. šok 34 C,5,,15,1,5 5 1 15 5 čas (h)

jabolčna kislina (g/l) vinska kislina (g/l) ocetna kislina (g/l) mlečna kislina (g/l) Temp. šok Temp. šok,8,7,6 18 C temp. šok 45 C x temp. šok 34 C 5 4,5 4 3,5 18 C temp. šok 45 C x temp. šok 34 C,5 3,4,5,3, 1,5 1,1,5 5 1 15 5 čas (h) 5 1 15 5 čas (h) Temp. šok Temp. šok 5 4,5 4 3,5 18 C temp. šok 45 C x temp. šok 34 C 4 3,5 3 18 C temp. šok 45 C x temp. šok 34 C 3,5,5 1,5 1 1,5 1,5,5 5 1 15 5 čas (h) 5 1 15 5 čas (h) Nastajanje ocetne in mlečne kisline, upadanje vinske in jabolčne kisline med fermentacijo pri 18ºC, minutnem temperaturnem šoku na vcepku pri 45ºC in dvakratnem 1 urnem temperaturnem šoku pri 34 ºC

Production of glycerol with various methods Ferm. Cont. 18ºC Single shock 8h/34ºC Oscilations 1h 18-5ºC Inoc. shock min/45ºc Glycerol (g/l) 6,81 9,4 11,6 1,14

Table 1. Biomass and metabolites of control fermentation and heat shocked inoculum. Products Control (18 C) (g l -1 ) Heath shocked inoculum ( min / 45 C) (g l -1 ) Biomass 6.7 ±. 7. ±. Red. sugars 6.5 ±.3.4 ±.9 Acetic acid.8 ±.6.3 ±. Succinic acid.63 ±.11.75 ±.1 Malic acid 3.3 ±.16 3.4 ±.17 Tartaric acid.5 ±.18. ±.9 Ethanol 84.7 ±.18 91.77 ±.93 Glycerol 6.81 ±,6 1.14 ±,6

- Butanol (g/l) Tartaric acid (g/l) 1 - Propanol (g/l) The Influence of Galvanic Field on Saccharomyces cerevisiae in Grape Must Fermentation 1,4 8,3 6,,,1 4,1,,, 4, 6, 8, 1, Fermentation time (days) 4 6 8 1 1 Fermentation time (days),,, 4, 6, 8, 1, Fermentation time (days) -Butanol Tartaric acid 1-Propanol

WINE FERMENTATION PROCESS CONTROL Redox control process regulation Temperature regulation CO pulses

etanol / (gl -1 ) Izbran profil redoks potenciala in poskus regulacije 3 5 decreasing T= 18 to 16 C injecting CO REG1 sladkor-reg etanol-reg IZBRAN E sladkor-izbran etanol-izbran 11 9 Eh / (mv), sladkor / (gl -1 ) 15 1 5-5 -1-15 increasing T = 16 - C end of CO injection 4 48 7 96 1 144 168 19 16 4 64 88 31 336 7 5 3 1-1 -3-5 - čas Fermentation fermentacije (h) / (h) -7

glukoza / (gl -1 ) fruktoza / (gl -1 ) etanol / (gl -1 ) glicerol / (gl -1 ) Glucose, fructose comsumtion and ethanol and glycerol production at redox controlled process 1 1 Ethanol 9 8 7 Glycerol 8 6 6 5 4 4 3 etanol-regulacija etanol-izbran 4 48 7 96 1 144 168 19 16 glicerol-regulacija 1 glicerol-izbran 4 48 7 96 1 144 168 19 16 Fermentation čas fermentacije (h) / (h) Fermentation čas fermentacije (h)/ (h) 1 1 Glucose glukoza-regulacija glukoza-izbrana 1 1 Fructose fruktoza-regulacija fruktoza-izbrana 8 8 6 6 4 4 4 48 7 96 1 144 168 19 16 4 Fermentation čas fermentacije (h) / (h) 4 48 7 96 1 144 168 19 16 4 Fermentation čas fermentacije (h)/ (h)

OTHER METHODS

REFLUX AROMA CONDENSERS

REFLUX AROMA CONDENSERS

LARGE SCALE REFLUX AROMA CONDENSERS

REFLUX AROMA CONDENSERS

:1 5:1 1:1 15:1 :1 5:1 3:1 35:1 4:1 45:1 5:1 55:1 6:1 65:1 7:1 T [ C], ph, kisik [%] prevodnost [mikros/cm], rh [mv] Crystalisation of potassium hidrogen tartarate in Sauvignon blanc wine at C with precipitation of KTH at on-line conductivity process control 8 1 14 14 6 4 Conductivity ph 1 8 1 1 8 Prevodnost 1 1 Temperature Oxygen 6 6 8-4 ph T 4 kisik rh prevodnost Temperatura Kisik ph 6 4-4 :5 :35 5:5 7:35 1:5 1:35 15:5 17:35 :5 :35 5:5-6 - èas [ura:min] Cooling T= C, termostation time 7 h Cooling on C with addition of 6g/l KHT conductivity on-line process control

:1 5:1 1:1 15:1 :1 5:1 3:1 35:1 4:1 45:1 5:1 55:1 6:1 65:1 7:1 T [ C], ph, kisik [%] prevodnost [mikros/cm], rh [mv] Crystalisation of potassium hidrogen tartarate in Malvasia wine at C with precipitation of KTH at on-line conductivity process control 1 14 1 16 1 1 1 14 8 6 4 - èas [ura:min] Cooling T= C, termostation time 7 ur 1 8 6 4 ph T kisik rh prevodnost 8 6 4 - Prevodnost Temperatura ph Kisik :5 5:5 1:5 15:5 :5 5:5 Cooling on C with addition of 6g/l KHT conductivity on-line process control 1 1 1 8 6 4

Sensor for SO measurement on-line measurement of free SO with modifyed CO electrode and sample acidification AS7 CONNECTOR ELECTRODE HOUSING MEMBRANE Potentiometric CO electrode

E [mv] Free SO measurement Red wines White wines 5-35 mg SO /l 5-5 mg SO /l -1 - -3-4 -5-6 y = 9,5Log(x) - 68, R =,979 1 1 1 log c SO [mg/l]

The Crew Marin BEROVIČ FKKT Ljubljana Aleksandra PIVEC ZRS Bistra Ptuj Tatjana KOŠMERL BF Ljubljana Mojmir WONDRA BF Ljubljana Andrej HOLOBAR ECHO Sl.Konjice Keith Racman Mettler Toledo Mateja Potočnik FKKT Ljubljana Jasna Štrus BF Ljubljana Štefan ČELAN ZRS Bistra Ptuj