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APPENDIX A. SUPPLEMENTARY DATA

Aspergilloglutamic peptidase (AGP) in combination with heat treatments

No AGP 120 10 mg/L AGP 100 50 mg/L AGP ) L / g 80 m (

n i e t

o 60 r p

d 40 i s e R 20

0 20 °C 60 °C 65 °C 70 °C 75 °C 80 °C -20 Heating temperature (1 minute each)

Figure S1. Protein content on the supernatants of 2009 Chardonnay juice samples heated at

20, 60, 65, 70, 75 and 80°C for 1 minute with different AGP dosages (0, 10, 50 mg/L). Heat treatments were performed in a heating block as follows: after filtration (at 0.22 µm), juice samples were prepared in 500 µL plastic tubes that were inserted into 2 mL tubes filled with water to favor heat transfer. A spare juice sample was used to measure the real temperature of samples during heating. The heating block was set 20 °C higher than the required temperature to allow a quick thermal equilibration of the samples. After heating, samples were cooled by immersing the sample tubes in ice for 3 minutes. Samples were than centrifuged (15000g,

4°C, 15 min) and supernatants used for protein analysis.

AGP worked at concentrations as low as 1 mg/L (data not shown), while 10 mg/L in combination with heating at 70°C yielded a protein reduction of 90%. SDS-PAGE analysis was performed on samples with 50 mg/L AGP heated at different temperatures (Figure S2).

Heating (1’) 20 60C 65C 70C 75C 80C kDa 0 50 0 50 0 50 0 50 0 50 0 50 250 AGP (mg/L) 150 100 75 Invertases 50 AGP 37

Chitinases 25 TLPs 20 15

LTP 10 Chardonnay juice, 4-12% gel, 1.5 mm

Figure S2. SDS-PAGE of samples heated at different temperatures in presence or absence of

50 mg/L AGP. The protein identity of bands was tentatively attributed (see names on the right).

The effect of heating alone on proteins can be seen when considering lanes where AGP was not added. In fact, heating alone caused a significant protein reduction from temperatures higher than the melting point of heat unstable wine proteins (65-70°C). As already observed by Sauvage, Bach, Moutounet, & Vernhet (2010) the first band to disappear is that around 35 kDa, possibly a β-glucanases. At 65°C the protein profiles started to be simplified, with bands decreasing in intensity or disappearing. The highest protein precipitation was observed with temperatures of 75-80°C. In the presence of 50 mg/L of AGP the protein reduction upon heating was greatly improved, with a generalised decrease in band intensities when compared to the controls. At 65°C a few faint bands were left and the profile does not change much by increasing the temperatures to 70°C or more. The AGP band almost disappeared from the

70°C treatments. It is of interest to note that the heat-stable wine proteins (LTPs, invertases), that is, the proteins not denatured by the heating, were resistant to the enzyme, suggesting that unfolded wine proteins are easily cleaved by the AGP.

A further preliminary experiment was conducted to determine the optimal heating temperature and AGP dosage for Chardonnay and Sauvignon Blanc juices used in small scale winemaking (Figure S3).

Sauvignon blanc Chardonnay 160 No AGP 15 mg/L AGP 25 mg/L AGP 140

) 120 L / g

m 100 (

t 80 n o c 60 n i e t o

r 40 P 20

0 20 65 70 75 20 65 70 75 20 65 70 75 Heating temperature (C, 1 minute)

Figure S3. Exploratory experiment on 2011 juices used for large scale experiments. Protein quantification (by EZQ) on samples heat treated for 1 minute at different temperatures in the presence of difference dosages of AGP. See Figure S4 for RP-HPLC chromatograms of the same samples. Data from Figure S3 (combined with those of Figure S1) confirmed that heating to at least

65°C was required for protein reduction. AGP added at 15 mg/L at 75°C yielded a protein reduction of 76% in Chardonnay and 83% in Sauvignon Blanc.

TLPs Chardonnay no AGP Sauvignon Blanc no AGP

) TLPs 400 U 400

A Chitinases ) m ( U

Chitinases n (

300 20 °C 0 300 m 1 n 2

0 t a 1 20 °C

2 e 200 65 °C t c

a 200 n

a e b c 70 °C r 65 °C n o

a 100 s b b

r 100 A o 70 °C s

b 75 °C A 0 75 °C 0 10 15 20 25 30 min 10 15 20 25 30 min

Chardonnay 15 mg/L AGP Sauvignon Blanc 15 mg/L AGP TLPs 400 Chitinases

400 TLPs ) U A m ) (

300 n m 300 (

Chitinases 0 1 m

2 20 °C n 20 °C

1 200 e 2

c 200 t 65 °C n a 65 °C

a b e r c o n 100 s

a 70 °C

70 °C b

b 100 r A o

s 75 °C b 75 °C A 0 0 10 15 20 25 30 min 10 15 20 25 30 min

Chardonnay 25 mg/L AGP Sauvignon Blanc 25 mg/L AGP TLPs Chitinases TLPs 400

400 ) U A ) m (

U A 300 m n m

Chitinases 300 (

20 °C 0 1 m 2

n 20 °C

t 0 a 1 200 2 e

t 65 °C

n 65 °C a

a e b r c o n 100 s a 70 °C b 70 °C

b 100 r A o s b 75 °C 75 °C A 0 0 10 15 20 25 30 min 10 15 20 25 30 min

Figure S4. RP-HPLC chromatograms of samples of Figure S3.

Figure S4 show the protein profiles by HPLC of juice samples treated at different temperatures and with different AGP addition rates. Sauvignon Blanc kDa MW C1 T2 T1 C2 kDa MW C1 T2 T1 C2 220 220 160 160 120 120 100 100 60 X X 70 F3 X X X 60 D10 X X 50 F11 H7 50 D11 40 G7 X 40 E7 X F12 G8 H8 D12 E8 30 30 H9 25 G1 G9 H3 F4 25 E1 F8 H10 E9 20 G2 G10 H4 E2 F9 E10 F5 20 G3 H5 E3 E11 F6 G11 15 G4 H11 E4 15 G12 H6 E12 G5 H1 H12 E5 F1 F7 F10 10 X 10 G6 X X E6 X X X

Figure S5. Soluble protein composition, as assessed by SDS-PAGE, of the finished

Sauvignon Blanc wine. Gels were performed in duplicate. Bands were excised and protein identities assessed by Peptide LC-MS/MS as shown in Table S4.

Chardonnay kDa MW C1 C2 T1 T2 kDa MW C1 T2 T1 C2

X X A1 D4 220 220 160 160 120 120 100 100 90 A2 X X X 90 B11 X X X 70 70 D1 X 60 B12 X X 60 A3 X X 50 X 50 C1 A4 B4 40 40 C2 A5 C8 X D5 B1 X C3 A6 X D6 30 B5 30 D2 25 C4 A11 B2 B6 25 A7 C9 D7 D3 20 C5 A12 B3 B7 20 A8 C10 D8 C11 B8 15 15

X C6 X X B9 A9 X D9 10 X X C7 X 10 A10 X X X

C12 B10

Figure S6. Soluble protein composition, as assessed by SDS-PAGE, of the finished Chardonnay wine. Gels were performed in duplicate. Bands were excised, named and protein identities assessed by Peptide LC-MS/MS as shown in Table S4. Table S1. Composition of wines presented for formal sensory analysis by triangle tests.

Glucose Free Total % Mali Lacti Bentonite Treatment T..A + SO2 SO2 Citric Tartari Succini Protei Haze Alc. pH c c requirement / Replicate (g/L) Fructos (ppm (ppm (g/L) c (g/L) c (g/L) n (g/L) (NTU) (v/v) (g/L) (g/L) (g/L) e (g/L) ) ) 12. 3.2 C1 Rep 1 6.3 1.3 20 110 0.30 3.37 2.08 1.41 0.32 125.0 1.9 80.5 6 9 12. 3.3 n.m. n.m. n.m. n.m. C1B Rep 2 5.6 1.3 25 115 n.m. 1.6 0 0.0 2 9 12. 3.2 T1 Rep 3 6.5 1.1 22 133 0.26 3.73 1.80 1.56 0.45 17.8 1.3 11.8 7 1 12. 3.2 C2 Rep 3 6.4 1.0 28 134 0.26 3.52 1.94 1.29 0.39 73.1 0.07 72.3 6 7 12. 3.3 T2 Rep 3 6.2 0.8 28 122 0.29 3.23 1.96 1.37 0.37 93.6 1.4 79.1 7 0 n.m. not measured

Table S2. Physicochemical parameters of Sauvignon Blanc wines after bottling.

Glu + Acetic Free Total Replicat 1 T.A. Tartari Succi Sauvignon Blanc Treatment Fru pH acid SO2 SO2 Citric Malic Lactic e (g/L)1 c nic (g/L)1 (g/L)1 (ppm)2 (ppm)2 1 1.3 3.29 6.3 0.38 20 110 0.30 3.37 2.08 1.41 0.32 Unheated juice C1 2 1.3 3.30 6.3 0.36 23 118 0.29 3.30 1.97 1.36 0.31 3 1.4 3.30 6.4 0.37 24 117 0.29 3.32 2.00 1.35 0.31 1 1.3 3.39 5.7 0.29 22 104 n.m. n.m. n.m. n.m. n.m. Unheated juice + bentonite C1B 2 1.3 3.39 5.6 0.28 25 115 n.m. n.m. n.m. n.m. n.m. 3 1.4 3.39 5.8 0.31 24 105 n.m. n.m. n.m. n.m. n.m. 1 0.8 3.30 6.2 0.36 25 122 0.29 3.23 1.95 1.38 0.37 Unheated juice + AGP T2 2 0.7 3.28 6.3 0.37 24 119 0.29 3.23 1.95 1.36 0.38 3 0.8 3.30 6.2 0.36 28 122 0.29 3.23 1.96 1.37 0.37 1 1.3 2.88 9.7 0.36 24 137 0.25 6.49 2.42 1.26 0.46 Heated juice + AGP T1 2 1.0 3.02 7.7 0.36 26 139 0.25 4.77 2.38 1.31 0.42 3 1.1 3.21 6.5 0.33 22 133 0.26 3.73 1.80 1.56 0.45 1 1.4 3.26 6.4 0.36 24 130 0.27 3.38 1.98 1.30 0.40 Heated juice C2 2 1.3 3.26 6.3 0.34 27 133 0.25 3.29 1.91 1.26 0.40 3 1.0 3.27 6.4 0.36 28 134 0.26 3.52 1.94 1.29 0.39 n.m., not measured 1. Glu/Fru analysis performed using enzymatic method, alcohol and acetic acid analysis performed by Winescan, pH and T.A. performed by auto-titrator. T.A. = Titratable Acidity, Glu + Fru = Glucose + Fructose. 2. Analysis performed by WIC Winemaking Services using aspiration method. 3. Differences in T.A. among replicates on treatment T1 were due to incomplete mixing of juice after acid adjustment.

* * Table S3. Mean CIELAB ∆E ab values for Sauvignon Blanc wines. Pairs with ∆E ab values

greater than 1 of are likely to be detected as different by the human eye.

* 1 Treatments ∆E ab Unheated (C1) vs Unheated + AGP (T2) 0.3 Unheated (C1) vs Heated + AGP (T1) 0.4 Unheated (C1) vs Heated (C2) 0.3 Unheated + AGP (T2) vs Heated + AGP (T1) 0.2 Unheated + AGP ( T2) vs Heated (C2) 0.1 Heated + AGP (T1) vs Heated (C2) 0.1 1 2 2 2 ΔEab * = [(L1–L2) + (a1-a2) + (b1–b2) ] Table S4. Identities of protein bands excised from gels of Figure S5 and S6.

d unique peptides 30 22 15 4 15 14 8 9 8 8 3 5 4 3 4

e s n e i l b

p total peptides 1610 187 1012 38 355 162 76 141 51 89 16 9 9 10 18

m m l l o a

A s c log(e) -380.4 -325.7 -212.6 -193.7 -166.2 -152.1 -118.9 -115.2 -91.3 -77.5 -65.8 -51.1 -35.1 -30.3 -29

Glycoside Glycoside Glycoside Plant lipid transfer Chitin recognition Papain family Plant lipid transfer pfam Thaumatin Thaumatin Thaumatin hydrolase hydrolase Barwin hydrolase protein protein cysteine protease protein

Glucan 1,3-beta- glucosidase Cysteine protease Vacuolar invertase Beta-1,3-glucanase (Saccharomyces Class IV chitinase Cp5 (Actinidia blast (Vitis vinifera GIN1) (Vitis riparia) cerevisiae) (Vitis vinifera ) deliciosa) log(e) total log(e) total log(e) total log(e) total log(e) total log(e) total log(e) total log(e) total log(e) total log(e) total log(e) total log(e) total log(e) total log(e) total log(e) total A1 no proteins identified A2 -26.7 5 -62.3 15.5 A3 -41.9 7 -60.8 20.5 A4 -46.5 8 -57.8 10 A5 -68 9 -60.5 12 -28.4 7.5 A6 -56.1 9 -79.6 13.5 -109.8 18 -80.7 13 A7 -150.2 27 -105.7 24 -69.3 15 A8 -240.5 71.5 -117.4 33 A9 -112.7 16 -41 6 -57.2 10.5 A10 -82.1 8 -38.4 6.5 -50.2 13 -37.6 6 A11 -151.1 20 -117.7 27.5 -55.9 16 A12 -216.7 60.5 -88.7 21 B1 -42.3 10 B2 -77.1 10.5 -69 15 -89.9 23 B3 -221.7 53.5 -117.7 17 B4 -69.8 10 -57.5 10 -19.9 3 -53.8 9.5 -31.2 4 B5 -70.1 8 -70.9 11 -35.7 6 -40.6 7.5 -33.4 4 B6 -99.6 12.5 -101.5 23 -59.1 21 B7 -217.8 76.5 -123.7 36 B8 -186.2 33.5 -77.9 9 B9 -88.2 11 -47.5 7 -66.6 13.5 -19.9 6 B10 no proteins identified B11 -91.3 11 -90.9 16 B12 no proteins identified C1 -50.9 8 C2 -86.7 12 -79.9 11 -30.6 7 C3 -117.5 16 -83.2 11 -56 9 -113 22.5 -79.3 13 C4 -125.6 15 -85.1 23.5 -59.4 23 C5 -263.8 108.5 -106.1 36 C6 -88.2 8 -37.9 6.5 -62.6 10 C7 -73 26.5 C8 -38.9 5 -48.1 9.5 -124.8 17 C9 -108.7 14 -87.9 23.5 -55.6 17 C10 -182.1 44.5 -91.7 18 C11 -108.7 28.5 -92.7 11 C12 no proteins identified D1 no proteins identified D2 -39.5 5 -59.8 9 -69.9 17.5 D3 -143.9 31.5 -67.2 9 D4 -61 9 D5 -113.1 15 -67 11.5 -18.8 4 D6 -82.3 6 -62.8 11.5 -48.6 7 D7 -91.8 15 -84.6 20.5 -59.7 16 D8 -249.7 102.5 -118.9 44 D9 -98.5 13 -66.7 10.5 D10 -28.6 8 D11 no proteins identified D12 no proteins identified E1 -107.6 14 -55.4 11 -82.6 15 -59.2 18.5 E2 -256.2 88.5 -309 57 -134.9 37 -56.9 6 E3 -111.7 15 -45.8 8 -66.7 10.5 E4 -41.5 7 E5 -32 4 -35.7 6 -65.4 18.5 E6 -72 18.5 E7 -113.1 16.5 E8 -49.8 6 -36.6 5.5 -49.7 9 -26.2 4 E9 -74.2 13.5 -76.1 12 -57.5 15 E10 -156.5 46 -133 27.5 E11 -106.6 28.5 -73.5 10 E12 -50.5 8 F1 -72.6 9 -44.4 6 F2 -45.6 6.5 F3 no proteins identified F4 -34.4 3 -35 7.5 -55.8 11 F5 -129.2 26.5 -79.2 14 F6 -60.8 10.5 -25.8 5 F7 -73.1 11 F8 -91.8 15 -64.6 12.5 -56.1 12 F9 -210.9 62.5 -114.1 23 F10 -56.8 10 F11 no proteins identified F12 no proteins identified G1 -102.1 16 -59.2 12.5 -98.1 14 -56.2 16 G2 -252.6 72.5 -266.4 41 -125.9 32 G3 -109.5 15 -29.8 7 -63.5 10.5 G4 -40.7 6.5 -44.1 6 G5 -43.6 6.5 -56.7 14 G6 -47.1 6 -46.4 14.5 G7 -66.2 7 -115 19.5 G8 -37.1 4 -33.6 5 G9 -126.4 24 -68.3 15.5 -58.5 14 G10 -199.1 47 -89 23.5 G11 -70.4 9 G12 -49.5 8 H1 -26.8 5 -32.7 5 H2 no proteins identified H3 -62.1 9.5 -68.1 9 H4 -22.6 4 -42.1 10 H5 -58.1 9 -46.4 8 H6 -38.4 5 H7 -36 5 H8 -34.5 5 -27.6 7.5 H9 -68.8 12 62.2 12.5 -50.5 11 H10 -244.6 65.5 -108.7 23 H11 no proteins identified H12 no proteins identified Table S5. Physicochemical parameters of Chardonnay wines after bottling.

Glu + % Acetic Free Total Replicat 1 T.A. Chardonnay Treatment Fru Alc. pH acid SO2 SO2 e (g/L)1 (g/L)1 (v/v)1 (g/L)1 (ppm)2 (ppm)2 3.3 1 5.7 13.3 6.1 0.57 24 115 8 3.3 Unheated juice C1 2 5.9 13.3 6.1 0.57 26 117 8 3.3 3 5.0 13.4 6.1 0.58 32 126 9 3.4 1 3.3 13.4 6.1 0.58 30 124 0 Unheated juice + AGP 3.4 2 3.7 13.4 6.1 0.56 34 127 T2 0 3.3 3 3.9 13.4 6.1 0.58 30 124 8 3.3 1 2.7 13.4 6.0 0.51 29 128 8 3.3 Heated juice + AGP T1 2 3.6 13.3 6.0 0.52 27 125 8 3.3 3 3.0 13.4 6.1 0.52 32 128 8 3.3 1 3.8 13.3 6.1 0.55 29 136 8 3.3 Heated juice C2 2 2.2 13.4 6.1 0.59 26 130 8 3.3 3 1.7 13.4 6.1 0.58 26 125 7 1. Glu/Fru analysis performed using enzymatic method, alcohol and acetic acid analysis performed by Winescan, pH and T.A. performed by auto-titrator. T.A. = Titratable Acidity, Glu + Fru = Glucose + Fructose. 2. Analysis performed by WIC Winemaking Services using aspiration method.

* Table S6. Mean CIELAB ∆E ab values for Chardonnay wines.

* 1 Treatments ∆E ab Unheated (C1) vs Unheated + AGP (T2) 0.3 Unheated (C1) vs Heated + AGP (T1) 0.4 Unheated (C1) vs Heated (C2) 0.2 Unheated + AGP (T2) vs Heated + AGP (T1) 0.6 Unheated + AGP ( T2) vs Heated (C2) 0.4 Heated + AGP (T1) vs Heated (C2) 0.2 1 2 2 2 ΔEab * = [(L1–L2) + (a1-a2) + (b1–b2) ]