Understanding Cap Extraction in Red Wine Fermentations
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1 Understanding Cap Extraction in Red Wine Fermentations Max Reichwage, Larry Lerno, Doug Adams, Ravi Ponangi, Cyd Yonker, Leanne Hearne, Anita Oberholster, and David Block Driving innovation in grape growing and winemaking
2 Understanding Cap Extraction in Red Wine Fermentors Motivation and key molecules Chemical gradients in red wine fermentors Effects of pumpover volume and frequency Effects of cap and must temperature on phenolic extraction
3 Cap Extraction in Red Wine Fermentors Add a second temp sensor Cap/Skins Color Monomeric Phenolics Tannin Polymeric Pigment Juice T
4 Closer look at the molecules extracted Polymeric flavan-3-ols (tannins) The most abundant class of phenolics in grapes Present in skins and seeds Anthocyanins Malvidin-3-glucoside is the predominant anthocyanin Found in the skin Hydroxycinnamates Ex: caftaric acid, caffeic acid, coumaric acid Found in the skin and pulp (Adams 2006)
5 Closer look at the molecules extracted Grape tannins-oligomers catechin (C), epicatechin (EC), epigallocatechin (EGC), and epicatechin gallate (ECG) Differences between skin and seed tannins Mean degree of polymerization (mdp) for skin tannin are ~30; seed tannins are ~10 (Souquet et al. 1996) Proportion of ECG units is different in seeds (~30%) and skins (~5%) (Cheynier et al. 2006) Hypothetical tannin tetramer (Adams 2006)
6 A molecular mechanism for cap extraction Epidermal Cell Layer Hypodermal Cells Mesocarp Cells Readsorption Polymeric Phenolics Release Reaction Structural Carbohydrate Monomeric and Polymeric Phenolics Vacuole (containing phenolics) Cell Nucleus All steps are likely a function of temperature and EtOH
7 Chemical gradients in red wine fermentations Driving innovation in grape growing and winemaking
8 Experimental Design: UC Davis/E&J Gallo Grapes: 2011 Cabernet Sauvignon from Lodi, CA 23.3 Brix, ph of 3.41, T.A. of 0.47 g/l Hand-picked, destemmed and crushed Inoculated with S. cerevisiae strain Lalvin D254 YAN adjusted to 300 ppm, addition of 50ppm SO 2 Pressed at dryness
9 Department of Viticulture and Enology Are there chemical gradients in red wine fermentations? Installed a curtain of 66 temperature sensors throughout the cross-section of a 2000 L tank 15 sample extraction points Fermented 2 Tons of Cabernet Sauvignon Pumped-over 1 tank volume 2x per day Peristaltic pump on catwalk for sample extraction
10 Analyses Performed 1. Phloroglucinolysis of Isolated Tannins - Tannin concentration, mdp, skin/seed contributions 2. RP-HPLC Phenolic Assay - Monomeric phenolic concentrations
11 Fermentation Profile Normalized y-axis : Brix (1.0 = 25 Brix), Free Anthocyanin (1.0 = 500 mg/l), Tannin (1.0 = 300 mg/l)
12 Results Free Anthocyanin Department of Viticulture and Enology hr 40 hr 48 hr 64 hr 72 hr 88 hr 96 hr 112 hr 120 hr 136 hr Units = mg/l
13 Free Anthocyanin Before and After A Pump-Over (Day 3) 8 am 10 am 11 am 12 pm 1 pm 2 pm 3 pm 4 pm Units = mg/l
14 Free Anthocyanin Before and After A Pump-Over (Day 5) 8 am 10 am 11 am 12 pm 1 pm 2 pm 3 pm 4 pm Units = mg/l
15 Tannin hr 40 hr 48 hr 64 hr 72 hr 88 hr 96 hr 112 hr 120 hr 136 hr Units = mg/l
16 Tannin Before and After A Pump-Over (Day 3) Units = mg/l 8 am 10 am 11 am 12 pm 1 pm 2 pm 3 pm 4 pm
17 Tannin Before and After A Pump-Over (Day 5) Units = mg/l 8 am 10 am 11 am 12 pm 1 pm 2 pm 3 pm 4 pm
18 Mean Degree of Polymerization (mdp) Department of Viticulture and Enology 40 hr 64 hr 88 hr 112 hr 136 hr possible mdp determined explanations: by 1. phloroglucinolysis Skin vs. seed tannin Early contribution in the fermentation - Initially, the larger mdp skin is higher in the cap tannins are extracted in the After 64 hours the mdp cap is ~13.5 throughout - Later in the fermentation, the fermentor the smaller seed tannins are Later extracted, in the bringing the fermentation mdp down the mdp decreases in 2. the Readsorption cap of larger tannins to the skin cell The error bars represent the pooled standard wall material deviation (considers 2 phloro - Larger reps tannin x 3 molecules SPE reps x 3 sampling points) have more reaction sites for hydrogen bonding
19 Percent Galloylation 40 hr 64 hr 88 hr 112 hr 136 hr Percent galloyation is the percentage of ECG subunits of tannins (determinged with phloroglucinolysis) % galloyation is greater in the seeds than the skins Increasing percent galloylation in the cap suggests increased extraction of tannins from the seeds
20 Phenolic Results - Catechin Units = mg/l 24 hr 40 hr 48 hr 64 hr 72 hr 88 hr 96 hr 112 hr 120 hr 136 hr Increases from ~8 mg/l to ~55 mg/l of catechin in the cap Relatively late to extract, could be a marker for seed extraction
21 Learnings from gradient studies Gradients in phenolics do exist Skin extraction is early, seed extraction is late Early cap samples may indicate final tannin level needs more work. Examining gradients in more detail may allow us to understand extraction at a more fundamental level
22 Effects of Pumpover Volume and Frequency Driving innovation in grape growing and winemaking
23 Fermentation treatments (2012) # $%"&#'"(# )*+,*-.$"'/ 0&/.$"'/ 0&/.1&(&2"'"(#.3"2*'" 04(#% : ;9<0 (=& >?.789.@45,'"A.;=-&B G 04(#%45.6C: ;9<0 (=& >?.C.@45,'"A.;=-&B 04(#%45.6;: ;9<0 (=& >?.;.@45,'"DA.;=-&B C ;7 ;7 H ; ;9 ;9 E E7 E7 GD.(""-"-.#4.'&*(#&*(.#"'/ F E9 E9 C ;7 ;9 0 ; ;9 E7 GD.(""-"-.#4.'&*(#&*(.#"'/ E E7 E9 All treatments are in triplicate. Measured phenolic profile at various time points in each fermentation
24 Department of Viticulture and Enology Wine production Grapes: 2012 Cabernet Sauvignon from Lodi, CA 24.3 Brix ph = 3.85 T.A. = 3.8 g/l (adjusted to 5.97 g/l) YAN adjusted to 300 ppm, addition of 50 ppm SO2 Inoculated with S. cerevisiae strain Lalvin D254 Pressed after 14 days 7-9 days extended maceration Sampling: AM & PM till dry, then AM only Fermentations performed in triplicate using Cypress/UC Davis Research Fermentors (TJs)
25 Using the TJ Fermentors Department of Viticulture and Enology
26 Effect of Pumpover Volume ' 560$"+0'#"$%&'5&%('!27&078$"927' #"$%&'"&%(')*+,-.' & % $ #!+',$-" #,$-" $,$-" &(" )*" #&&" #)$" $& $((" %%*" /%*0')123$4.' 560$"+0'80&:%7'!27&078$"927' * 80&:%7')*+,-.' ' & % $ #!+',$-" #,$-" $,$-" &(" )*" #&&" #)$" $& $((" %%*" /%*0')123$4.'
27 Effect of Pumpover Volume 6#147'1)!7"#0809$%$&$'"(:35081) ;39:19<47=39) #$%$&$'"()*+',-.) %! $# $! # &'" ()" $&&" $(%" %& %''" **)" /0+1)*23(45.)!+#,%-" $,%-" %,%-"
28 Examining pumpover frequency # $%"&#'"(# )*+,*-.$"'/ 0&/.$"'/ 0&/.1&(&2"'"(#.3"2*'" :;.<=./"%.-&> E :;.5=./"%.-&>? 56 70?.9:;.4=./"%.-&> 4 4.-&>.0:;-.!:&AB :;.5=./"%.-&> F 5?.-&>.0:;-.!:&AB C.-&>.0:;-.!:&AB :;.5=./"%.-&>? 4D.-&>.0:;-.!:&AB :;.5=./"%.-&> For 2013, pressed all batches at 8 days.
29 Pumpover frequency does not affect phenolic extraction '#" #"$%&'"&%('&25&056$"725'"6'(%80$056'93*9:2;0$'<$0=305&>' ' #"$%&'"&%(')*+,-.' &#" & %#" % $#" $ #",-$.",-%.",-'.",-+." $#" & '#" ( )#" * $!#" $% $&#" $# $(#" $+ $*#" /%*0')123$4.' ##" # '#" ' #$%&'()%1(%$(#2"41()"#)5'6$2$(#)78+791:$2);2$<8$(%=) #$%&'()*+,-./) &#" & %#" % $#" $ #" $#" & '#" ( )#" * $!#" $% $&#" $# $(#" $+ $*#" 0'+$)*&123/),-$.",-%.",-'.",-+." RP-HPLC
30 Pumpover frequency does not affect phenolic extraction '% #$%&%'()(*#+,-.%&/0,-',/'98":-'0"90&%;/8/'90<+2<(-$/80=8/>+/',?0 '$ #$%&%'()(*#+,-.%&/012*3450 '# '! & % $,-'.",-#.",-$.",-&." # '(" ) $(" % *(" + '!(" '# ')(" '( '%(" '& '+(" 6%2/ '$ ##$#%2.#2,#30"4.#%"3%5$6,0,#3%7/'78.9,0%:0,;/,#2<% '# ##$#%&'()*+% '! & % $,-'.",-#.",-$.",-&." # '(" ) $(" % *(" + '!(" '# ')(" '( '%(" '& '+("!$',%&-./01+% RP-HPLC
31 No change in color 8$9$%*:)'#4;<*:7%4'=*>)%5)';&?$'* #$%"&'() *+,-./0-./1-.2* +" *" )" (" '" &" %" $" #" #'" % &'" ( )'" + #!'" #$ #%'" #' #('" #* #+'" 345)*+6$7%2*,-#.",-$.",-&.",-*." #$%"&'()* +,-./0-./1-.2* +" *" )" (" '" &" %" $" #" 8$9$%*:)'#4;<*$>*D'4#6):*E4')* &* &* &* UV-VIS
32 #$%!&'()*)+,-,"'./01)*2%23.)(&'2+41% )! (! '! &! %! $! #! Department of Viticulture and Enology No effect on anthocyanin or tannin extraction :!0.+4%0;%:+470/<&+)+1%*.8)+"%;28!2+4&=0+" #'" % &'" ( )'" * #!'" #$ #%'" #' #('" #+ #*'" 5)!2%670.89%,-#.",-$.",-&.",-+." #$%!&'()*)+,-,"'./01)*2%23.)(&'2+41% (! '! &! %! $! #! :+470/<&+)+1%0;%>+)172*%?)+2% &% &%,-#.",-$.",-&.",-+." &% &% #$%/&42/7)+%23.)(&'2+41% :!0.+4%0;%5&++)+1%*.8)+"%;28!2+4&=0+% '! &' &! %' %! $' $! #' #! ' #'" % &'" ( )'" * #!'" #$ #%'" #' #('" #+ #*'" 5)!2%670.89%,-#.",-$.",-&.",-+." #$%/&42/7)+%23.)(&'2+41% '! &' &! %' %! $' $! #' #! ' 5&++)+1%0;%>+)172*%?)+2% &% &% &% &%,-#.",-$.",-&.",-+." UV-VIS/AH Correlation
33 No effect on phenolic profile of finished wine #! + * #$%&'(")$*+,-" ) ( ' & % $ ;<=#">?4" ;<=$">?4:" ;<=&">?4:" ;<=*">?4:" #,-../0"-0/1" /6" 78/0-3405/6" 9-66/6:" &#" & %#" #$%&'(")$*+,-" % $#" $ 674$"852" 674%"8529" 674:"8529" 674;"8529" #" '()(*+,"(,+-" './0(*+," '(12+,"(,+-" 34,./5(*+,"(,+-" RP-HPLC
34 No effect on origin of tannins +)-.&#'$&"/)0#12)345%3) +)-.&#'$&"/)0#12)3""/3) )#" (#" )#" (#" #$"%&'(")*+,) '#" &#" %#" #$"%&'(")*+,) '#" &#" %#" $#" $#" #" *+$," *+%," *+'," *+-," #" *+$," *+%," *+'," *+-," Phloroglucinolysis
35 Learnings from pumpover studies Pumpover volume does not make a difference in phenolic extraction (for volumes tested) Pumpover frequency does not make a difference in phenolic extraction
36 Effects of Cap and Must Temperature on Phenolic Extraction Driving innovation in grape growing and winemaking
37 Fermentation treatments (2012) # $%"&#'"(# )*+,*-.$"'/ 0&/.$"'/ 0&/.1&(&2"'"(#.3"2*'" 04(#% : ;9<0 (=& >?.789.@45,'"A.;=-&B G 04(#%45.6C: ;9<0 (=& >?.C.@45,'"A.;=-&B 04(#%45.6;: ;9<0 (=& >?.;.@45,'"DA.;=-&B C ;7 ;7 H ; ;9 ;9 E E7 E7 GD.(""-"-.#4.'&*(#&*(.#"'/ F E9 E9 C ;7 ;9 0 ; ;9 E7 GD.(""-"-.#4.'&*(#&*(.#"'/ E E7 E9 All treatments are in triplicate. Measured phenolic profile at various time points in each fermentation
38 Department of Viticulture and Enology TJ Fermentors,-./&."4&#$34&4%*5$& 6+1#&#$34&4%*5$& :";<$#&#$34& 4%*5$& 0$(1'#2& 3$#$%& #$%&*+#)$#& #$%&'()$#& /;%$$(& 7+34& 18"9&
39 Effect of Cap and Liquid Temperature ' 560$"+0'#"$%&'5&%('!27&078$"927' #"$%&'"&%(')*+,-.' & % $ # &(" )*" #&&" #)$" $& $((" %%*" /%*0')123$4.' $!+$!," $'+$'," %!+%!," %'+%'," 560$"+0'80&:%7'!27&078$"927' 80&:%7')*+,-.' - * ' & % $ # &(" )*" #&&" #)$" $& $((" %%*" /%*0')123$4.' $!+$!," $'+$'," %!+%!," %'+%',"
40 The effect of cap and liquid temperature 23,0"(,%##$#%4.#5,#60"7.#% ##$#%&'()*+% '# '! &# &! %# %! $# $! # '(" )*" $''" $)%" %' %((" &&*"!$',%&-./01+% %!+%!," %#+%#," &!+&!," &#+&#," 8$(',#69%8.:;9%&'()*+% # ' & % $ 23,0"(,%8$(',#6,<%8.:;',0% 4.#5,#60"7.#% '(" )*" $''" $)%" %' %((" &&*"!$',%&-./01+% %!+%!," %#+%#," &!+&!," &#+&#,"
41 The effect of cap and liquid temperature 6#147'1)!7"#0809$%$&$'"(:35081) ;39:19<47=39) #$%$&$'"()*+',-.) %! $# $! # &'" ()" $&&" $(%" %& %''" **)" /0+1)*23(45.) %!+%!," %#+%#," *!+*!," *#+*#,"
42 Is the main driver for extraction the liquid temperature or cap temperature? ) 67$4",$)#$%&'()!2(%$(#4"82() ( ' #$%&'()*+,-./) & % $ $!,$!-" $',$'-" %!,%!-" %',%'-" # &*" +(" #&&" #+$" $& $**" %%(" 0'+$)*12345/)
43 Is the main driver for extraction the liquid temperature or cap temperature? ) 67$4",$)#$%&'()!2(%$(#4"82() ( #$%&'()*+,-./) ' & % $ $!,$!-" $',$'-" %!,%!-" %',%'-" $!,$'-" $',%!-" %!,%'-" # &*" +(" #&&" #+$" $& $**" %%(" 0'+$)*12345/)
44 Liquid temperature or cap temperature? '# 23,0"(,%##$#%4.#5,#60"7.#% '! &# &! ##$#%&'()*+% %# %! $# %!+%!," %#+%#," &!+&!," &#+&#," $! # '(" )*" $''" $)%" %' %((" &&*"!$',%&-./01+%
45 Liquid temperature or cap temerpature? '# 23,0"(,%##$#%4.#5,#60"7.#% '! &# ##$#%&'()*+% &! %# %! $# $! %!+%!," %#+%#," &!+&!," &#+&#," %!+%#," %#+&!," &!+&#," # '(" )*" $''" $)%" %' %((" &&*"!$',%&-./01+%
46 a) b) c) d)
47 a) b) c) d)
48 Learnings from cap and liquid temperature studies Temperature affects how rapidly skins are extracted (not necessarily how much is extracted) Temperature definitely affects seed extraction Liquid temperature seems to be more important than cap temperature for extraction
49 Summary Chemical gradients were observed for a number of important compounds Extraction of compounds located in the skin appears to occur early in fermentation Could early cap tannin be a marker for final tannin levels? Extraction of compounds located in the seeds appears to occur later in the fermentation and is more temperature dependent Liquid temperature seems to have more effect than pumpover volume on extraction Understanding the mechanism of extraction will allow better manipulation of phenolic profiles
50 Acknowledgements Dr. Roger Boulton Chik Brenneman Paul Green Tim Jones Dr. Cary Doyle John Schadt Tom Bell (Bainer Hall Machine Shop) Department of Viticulture and Enology Collin Chew Dustin Owens The Adams Lab The Ebeler Lab E&J Gallo Winery: Dr. Leanne Hearne Dr. Ravi Ponagi Dr. Tom Pugh Dr. Nick Dokoozlian Cynthia Yonker Winery Interns: Daina, Jeff, Ben, Laura, and Quinton Funding Sources: - E&J Gallo Winery - UC Davis Federal Block Grant - Harry Baccigaluppi Scholarship - Brad Webb Memorial Scholarship - Wine Spectator Scholarship
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