VINOLOK Closure Evaluation. Stage 2: Closure Performance Benchmarking 24-month Report. 14 October, AWRI Report

Transcription

1 WRI Report VINOLOK Evaluation Stage 2: Performance Benchmarking 24-month Report uthors: Kieran Hirlam Neil Scrimgeour 14 October, 2016 Page 1 of 44

2 Disclaimer This document has been prepared by The ustralian Wine Research Institute ("the WRI") for a specific purpose and is intended to be used solely for that purpose and unless expressly provided otherwise does not constitute professional, expert or other advice. The information contained within this document ("Information") is based upon sources, experimentation and methodology which at the time of preparing this document the WRI believed to be reasonably reliable and the WRI takes no responsibility for ensuring the accuracy of the Information subsequent to this date. No representation, warranty or undertaking is given or made by the WRI as to the accuracy or reliability of any opinions, conclusions, recommendations or other information contained herein except as expressly provided within this document. No person should act or fail to act on the basis of the Information alone without prior assessment and verification of the accuracy of the Information. To the extent permitted by law and except as expressly provided to the contrary in this document all warranties whether express, implied, statutory or otherwise, relating in any way to the Information are expressly excluded and the WRI, its officer, employees and contractors shall not be liable (whether in contract, tort, under any statute or otherwise) for loss or damage of any kind (including direct, indirect and consequential loss and damage of business revenue, loss or profits, failure to realise expected profits or savings or other commercial or economic loss of any kind), however arising out of or in any way related to the Information, or the act, failure, omission or delay in the completion or delivery of the Information. In the event that any legislation or rule of law implies any condition, warranty or liability with respect to the WRI or the Information, the WRI s liability for breach of any condition, warranty or liability shall be limited, at the option of the WRI, to the re-supply of that Information; the cost of acquiring equivalent Information or the payment of the cost of having the Information re-supplied. The Information is confidential and may be legally privileged. If you are not the intended recipient of the Information, please immediately notify the WRI and destroy the Information. Unless expressly provided in this document, the WRI retains ownership of the copyright in the Information and no part of the Information may be reproduced or copied in any form or by any means without the prior written consent of the WRI. The Information must not be used in a misleading, deceptive, defamatory or inaccurate manner or in any way that may otherwise be prejudicial to the WRI, including without limitation, in order to imply that the WRI has endorsed a particular product or service.

3 1 Introduction The ustralian Wine Research Institute (WRI) was engaged to carry out a closure benchmarking trial for a newly developed Vinolok low-top closure. series of physical, chemical and sensory tests have been applied to wines sealed with the new (18.5mm) closure, and performance compared with the older (18.2mm) design Vinolok closure, as well as screw-cap (Saran/tin) and natural cork closures at regular intervals over a period of 36 months. This report includes an updated summary of all of the analysis carried out on the wines up to and including 24 months postbottling.

4 t Bottling 3-Month 9-Month 12-Month 18-Month 24-Month Commercial in Confidence 2 Materials & Methods Bottling and initial chemical tests were carried out during pril month analysis was conducted in pril Table 1 provides a summary of the testing schedule: Table 1: Testing Schedule Summary nalytical Test Basic Chemical ttributes Free and Total SO 2 Total Packaged Oxygen (TPO) Wine Colour Oxygen Transmission Rate (OTR) Low Molecular Weight Sulfides (LMWS) Sensory nalysis (aroma & palate) 2.1 Wine Storage ll bottled wine samples have been stored at the Wine Innovation Cluster Winery Bottle Store on site at Waite Campus, Urrbrae. The storage conditions were in darkness at a temperature of approximately 17 o C and 55 % relative humidity. 2.2 Benchmarking Chemical nalyses ll chemical analyses were performed by WRI Commercial Services NT accredited (ISO certified) wine laboratory. ll chemical analyses were performed by trained staff in accordance with NT accredited quality assurance measures including standards, blanks, duplicates and control samples. The quality control measures were required to meet established criteria before acceptance of the analytical data. ll samples were analysed in a randomised run order, to mitigate the risk of instrument or sample drift across the sample set influencing the results. The uncertainty of measurement (UOM) inherent in the analytical data is shown in brackets below. WineScan was used to measure the following basic chemical attributes of three replicate samples: % lcohol (± 0.1% v/v) ph (± 0.05) Titratable acidity (± 0.1 g/l) Volatile acidity (± 0.04 g/l) Glucose/Fructose (± 0.3 g/l) Specific Gravity (± )

5 Free and total SO 2 was measured on three replicate samples using Flow Injection nalysis (FI, Lachat; UOM ± 3 mg/l). White and red wine colour measurement was performed on three replicate samples utilising absorbance measurements at 280, 320 and 420nm on a Varian UV/Visible spectrophotometer. This method utilises standardised wine ph and alcohol concentration to enable the determination of the following parameters: Hue (± 5%) Wine colour density (± 5% a.u.) Chemical ge 1 (± 5%) Chemical ge 2 (± 5%) Free anthocyanins (± 10% mg/l) Pigmented Tannin (± 5% a.u.) Total phenolics (± 10% a.u.) Total pigment (± 10% a.u.) LMWS compound analysis was conducted on three replicate samples using static headspace sampling combined with an gilent gas chromatograph fitted with a sulfur chemi-luminescence detector (GC-SCD). Compounds analysed include: Hydrogen sulfide Methanethiol Ethanethiol Dimethyl sulfide Carbon disulfide Diethyl sulfide Methyl thioacetate Dimethyl disulfide Ethyl thioacetate Diethyl disulfide OTR OTR testing was performed using the WRI s proprietary wet-otr method to ascertain OTR values nondestructively. This method utilises a customised non-reactive housing fitted with a PreSens Pst3 oxygen sensor. This is used to encase the area surrounding the closure, therefore creating a sealed reservoir of air external to the closure. The oxygen concentration within the housing is continually monitored at constant temperature (17 C). Measurements are used to calculate the rate of oxygen transmission through the samples. Three replicates of each closure were trialled for a period of 4 weeks, stored upright and in darkness at 17 C Sensory Evaluation Wines were informally assessed by the WRI Sensory Team prior to the beginning of the sensory panel training. panel of twelve assessors (six males, six females) with an average age of 49 years (SD = 14.1) was convened to evaluate the white wines and a panel of ten assessors (five males, five females) with an average age of 53 years (SD = 12.0) was convened to evaluate the red wines. ll of whom are part of the WRI trained descriptive analysis panel.

6 The white wine set was studied initially, followed by the red wine set. The same procedure was followed for both sets. ssessors attended one training session to determine whether the attribute list previously used to evaluate the wines after 12 months in bottle still contained appropriate descriptors for rating in the formal sessions. During this session the assessors assessed all of the wines from the study. Wines were assessed by appearance, aroma and palate. Standards for aroma attributes were presented and discussed and these standards were also available during the booth practice session and the formal assessment sessions. Following the training session, tasters participated in a practice session in the sensory booths under the same conditions as those for the formal sessions. fter the practice session, any terms which needed adjustment were discussed and the final list of terms determined. For the white wine formal sessions this list was refined to include one ppearance term, fourteen roma terms (thirteen defined and Other ) and thirteen palate terms (twelve defined and Other ). For the Red wine formal sessions there was one ppearance term, thirteen roma terms (twelve defined and one Other ), and fifteen Palate terms (fourteen defined and one Other ). These attributes, definitions/synonyms and standards provided are shown in Table 2 and 3, including only those attributes which were included in the final attributes list. Table 2: ttributes, definitions and reference standards evaluated by panellists in formal sessions for the white wine samples ttribute Definition/Synonyms ppearance Yellow Colour Intensity Intensity of the colour yellow in the sample roma Overall fruit intensity aroma Intensity of the fruit aromas Tropical Intensity of the aroma of tropical; passionfruit, pineapple, melon and mango. Stonefruit Intensity of the aroma of stonefruits: peach, apricot, nectarine both fresh and dried. Citrus Intensity of the aroma of citrus fruits: lemon, lime, grapefruit and orange. Floral Intensity of the aroma of flowers: violets and blossoms, musk. Green Intensity of the aroma of green leaves, stalks, green capsicum and cucumber and herbal. Dry Grass Intensity of the aroma of dried grass, earthy and dusty. Vegetal Intensity of the aroma of various vegetables: cooked vegetables such as asparagus and green beans, water vegetables have been cooked in, drain. Box Hedge Intensity of the aroma of box hedge. Cardboard Intensity of the aroma of cardboard, bread, yeast. Flint Intensity of the aroma of flint, wet stones, metals, toast. Sweaty/Cheesy Intensity of the aroma of sweat, cheese, blue cheese, cheddar cheese, body odour, sour milk, Pungent Intensity of the aroma and effect of alcohol. Palate Overall fruit intensity palate Intensity of fruit flavours Tropical Fruit Intensity of the flavour of tropical fruits: pineapple, passionfruit, melon, mango. Stonefruit Intensity of the flavour of stonefruits: peach, apricot, nectarine. Citrus Intensity of the flavour of citrus fruits: lemon, lime, orange, grapefruit. Green Intensity of the flavour of green stalks, green gooseberries, green leaves, grass and green Sweet Intensity of the taste of sucrose. Viscosity cid Hotness The perception of the body, weight or thickness of the wine in the mouth. Low=watery, thin mouth feel. High=oily, thick mouth feel. Intensity of acid taste in the mouth including aftertaste. The intensity of alcohol hotness perceived in the mouth, after expectoration and the associated burning sensation. Low = warm; High = hot.

7 stringency Bitter Fruit T The drying and mouth-puckering sensation in the mouth. Low=coating teeth; Medium=mouth coating & drying; High=puckering, lasting astringency. The intensity of bitter taste perceived in the mouth, or after expectoration. The lingering fruit flavour perceived in the mouth after expectorating. Table 3: ttributes, definitions and reference standards evaluated by panellists in formal sessions for the red wine samples ttribute ppearance Opacity roma Overall fruit aroma intensity Red fruits Dark fruits Confection Floral Green Cooked Vegetables Spice Vanilla Earthy Woody Pungent Palate Overall fruit intensity Red Fruit Dark Fruit Green Vanilla Spice Woody Sweet Viscosity cid Hotness stringency Bitter Fruit T Definition/Synonyms The degree to which light is not allowed to pass through a sample Intensity of the fruit aromas in the sample Intensity of the aroma of red fruits and berries: raspberries, strawberries, cranberries Intensity of the aroma of dark fruits and berries: blackberries, plums, cherries, black currants Intensity of the aroma of confection: raspberry lollies, musk lollies Intensity of the aroma of flowers: violets, roses Intensity of the aroma of green stalks, leaves, grass, green beans Intensity of the aroma of various vegetables, cooked vegetables, water vegetables have been cooked in, drain Intensity of the aromas of various sweet spices: cinnamon, cloves, mixed spice, cardamom. Intensity of the aroma of vanilla Intensity of the aroma of wet earth, organic matter, compost, mushrooms, mud and dust Intensity of the aroma of wood, oak, pencil shavings Intensity of the aroma and effect of alcohol Intensity of fruit flavours in the sample Intensity of the flavour of red fruits and berries: raspberries, strawberries, cranberries Intensity of the flavour of various dark fruits: blackberries, currants, plums, cherries Intensity of the flavour of green stalks, green capsicum, green bean Intensity of the flavour of vanilla Intensity of the flavour of various spices: cinnamon, cloves, mixed spice, cardamom Intensity of the flavour of wood, oak Intensity of the taste of sucrose The perception of the body, weight or thickness of the wine in the mouth. Low=watery, thin mouth feel. High=oily, thick mouth feel. Intensity of acid taste in the mouth including aftertaste The intensity of alcohol hotness perceived in the mouth, after expectoration and the associated burning sensation. Low = warm; High = hot, burning. The drying and mouth-puckering sensation in the mouth. Low=coating teeth; Medium=mouth coating & drying; High=puckering, lasting astringency. The intensity of bitter taste perceived in the mouth, or after expectoration. The lingering fruit flavour perceived in the mouth after expectorating.

8 The intensity of each attribute was rated using an unstructured 15 cm line scale from 0 to 10, with indented anchor points of low and high placed at 10% and 90% respectively. Data was acquired using Fizz sensory software (version 2.46, Biosystemes, Couternon, France). Panel performance was assessed using Fizz, Senstools (OP&P, The Netherlands) and PanelCheck (Matforsk) software, and included analysis of variance for the effect of judge and presentation replicate and their interactions, degree of agreement with the panel mean and degree of discrimination across samples. For the white wine study, eleven of the twelve judges were found to be performing to an acceptable standard leaving the panel consisting of five males and six females with an average age of 51 (SD=12.9). Eight of the ten panellists in the red wine study were found to be performing to an acceptable standard, leaving the panel consisting of four males and four females with an average age of 57 (SD=8) Method of statistical analysis ll data was analysed by one-way NOV techniques combined with Tukey s multiple comparisons test using the statistical package GraphPad Prism 6.0 (GraphPad Software, Inc.). Statistical significance has been defined at the 95% confidence interval. With regard to sensory evaluation, analysis of variance (NOV) was carried out using Minitab (Minitab Inc., Sydney, NSW). The effects of closure (C), judge (J), replicate (R) and their two-way interactions were assessed, treating judge as a random effect. Following NOV, Fisher s least significant difference (LSD) value was calculated (P=0.05).

9 Free SO2 (mg/l) Free SO2 (mg/l) Commercial in Confidence 3. Results and Discussion The results are presented as follows: Free and Total SO 2 levels Wine Colour Low Molecular Weight Sulfides (LMWS) Sensory analysis 3.1 Free and Total SO2 Free and total SO 2 trends for white wine over the initial 24 months of the trial are presented below in Figure 1 and Figure 2, respectively. Complete data is provided in ppendix B Time (Months) Natural Cork Ref 1 Screw Cap (Saran/Tin) Vinolok 18.2 Vinolok B B B 5 0 Screw Cap (Saran/Tin) Vinolok 18.5 Vinolok 18.2 Natural Cork Ref 1 Figure 1a (top) White wine free SO 2 trends over the 24 months since bottling & 1b (bottom) Free SO 2 results 24 months since bottling shown in descending order. Error bars indicate standard deviations. Lettering denotes significant difference between closure varaints (i.e. vs. B: significantly different / B: neither significantly different to closures denoted with or with B etc.)

10 Total SO2 (mg/l) Total SO2 (mg/l) Commercial in Confidence Time (Months) Natural Cork Ref 1 Screw Cap (Saran/Tin) Vinolok 18.2 Vinolok Screw Cap (Saran/Tin) B B Vinolok 18.5 Vinolok 18.2 Natural Cork Ref 1 Figure 2: White wine total SO 2 trends over the 24 months since bottling & 1b (bottom) Total SO 2 results 24 months since bottling shown in descending order. Error bars indicate standard deviations. Lettering denotes significant difference between closure variants (i.e. vs. B: significantly different / B: neither significantly different to closures denoted with or with B etc.) The trends observed within the white wine SO 2 data remain consistent with what has been observed over the duration of the trial. There has been a noticeable plateau in free and total SO 2 values for the white wine since the 18month time point; however, this effect has been observed in similar closure trials of this nature. Significant differences between closures are still present at 24 months, with screw caps retaining the highest amount of SO 2 and cork showing the biggest drop. No significant differences are evident between the two Vinolok closures for free and total SO 2 in the white wine.

11 Free SO2 (mg/l) Free SO2 (mg/l) Commercial in Confidence Free and total SO 2 trends for the red wine over the initial 24 months of the trial are presented below in Figure 3 and Figure 4, respectively. Complete data for the selected closure set is provided in ppendix B Time (Months) Natural Cork Ref 1 Screw Cap (Saran/Tin) Vinolok 18.2 Vinolok B BC C 5 0 Screw Cap (Saran/Tin) Vinolok 18.2 Vinolok 18.5 Natural Cork Ref 1 Figure 3: Red wine free SO 2 trends over the 24 months since bottling & 1b (bottom) Free SO 2 results 24 months since bottling shown in descending order. Error bars indicate standard deviations. Lettering denotes significant difference between closure variants (i.e. vs. B: significantly different / B: neither significantly different to closures denoted with or with B etc.)

12 Total SO2 (mg/l) Total SO2 (mg/l) Commercial in Confidence Time (Months) Natural Cork Ref 1 Screw Cap (Saran/Tin) Vinolok 18.2 Vinolok Screw Cap (Saran/Tin) B B B Vinolok 18.5 Vinolok 18.2 Natural Cork Ref 1 Figure 4: Red wine total SO 2 trends over the 24 months since bottling & 1b (bottom) Total SO 2 results 24 months since bottling shown in descending order. Error bars indicate standard deviations. Lettering denotes significant difference between closure variants (i.e. vs. B: significantly different / B: neither significantly different to closures denoted with or with B etc.) Similar to the white wine data, the trends observed within the red wine SO 2 data remain consistent with what has been observed over the duration of the trial. Wines have shown limited SO 2 depletion since the 18-month timepoint. The slight increases shown for samples under screw cap could be attributed to sampling variability and/or analytical measurement error (uncertainty ±3mg/L). Significant differences can still be observed between closures with data showing the red wine has experienced the biggest free SO 2 drop under the cork closure and the screw cap the smallest over 24 months. The performances of

13 the Vinolok closures continue to remain between these two reference closures with no significant differences between the pair.

14 Total Pigment (a.u.) Total Pigment (a.u.) Commercial in Confidence 3.2 Wine Colour The differences in the wine colour parameters 24-months post bottling were still small as seen throughout the trial, with only the total pigment showing a significant difference between the cork samples and the two Vinolok samples. The total pigment results are presented in Figure 5 below. ppendix C provides a summary of all red wine colour data up to and including the 24-month analysis point Storage (Months) Natural Cork Ref 1 Screw Cap (Saran/Tin) Vinolok 18.2 Vinolok B B Vinolok 18.2 Vinolok 18.5 Screw Cap (Saran/Tin) Natural Cork Ref 1 Figure 5a (top) Wine colour (Total Pigment) trends over the 24 months since bottling & 5b (bottom) Total Pigment results 24 months since bottling shown in descending order. Error bars indicate standard deviations.

15 OD420nm (a.u.) OD420nm (a.u.) Commercial in Confidence Similar to that observed at the 18-month time point, there are very few differences in colour attributes for the white wine, due to closure impact, at the 24-month time point. pparent differences and separation continue to exist for optical density measured at 420nm (browning) as seen at the 18-month time point. Significant differences exist between the two Vinolok samples, with the 18.5mm closure displaying an apparent decline over the last sixmonths. This is likely a result of sample to sample variation. The optical density 420nm results are presented in Figure 6 below. ppendix D provides a summary of all white wine colour data up to and including the 24-month analysis point Storage (Months) Natural Cork Ref 1 Screw Cap (Saran/Tin) Vinolok 18.2 Vinolok B C C Natural Cork Ref 1 Vinolok 18.2 Vinolok 18.5 Screw Cap (Saran/Tin) Figure 6a (top) OD420 trends over the 24 months since bottling & 5b (bottom) OD420 results 24 months since bottling shown in descending order. Error bars indicate standard deviations.

16 Carbon Disulfide (µg/l) Commercial in Confidence LMWS compounds Several LMWS compounds were detected within the wine samples, including dimethyl sulfide (DMS), carbon disulfide (CS 2), hydrogen sulfide (H 2S), methanethiol (MeSH) and methyl thioacetate (MT). The bottled white wine contained DMS and MeSH present at levels greater than the sensory perception thresholds for all samples, whereas the red wine had three of the five listed compounds present at levels greater than the threshold levels for all samples. t the 24-month time point, only one significant difference was observed for the detected LMWS compounds in the red wine (CS 2), although the levels present under all closures are well below the sensory perception threshold (38µg/L). Methanethiol concentrations increased marginally under all closures and the small, yet significant, differences at the 12-month time point became insignificant at the 24-month time-point. Figure 7 shows the development of CS 2 in red wine over 24 months in bottle. clear separation has formed between the two reference closures and the Vinolok closures, with concentrations in the order of ~2µg/L higher under the reference closures (only significantly different between the screw cap and Vinolok closures). The concentrations present for most of the detected LMWS compounds at the 24-month time point are either just above (H 2S and MeSH) or well below (CS 2 and MT) the sensory perception thresholds of these compounds in wine. Dimethyl sulfide (DMS) has continued to develop under all closures in the red wine, significantly surpassing the aroma detection threshold (25µg/L); however, levels remain relatively low comparative to those seen in commercial wines (0-980 µg/l) Month 9 Month 12 Month 24 Month 1 0 Natural Cork Ref 1 Screw Cap (Saran/Tin) Vinolok 18.2 Vinolok 18.5 Figure 7: LMWS (Carbon Disulfide) analysis up to 24-months post bottling in red wine samples. MeSH and H 2S were the two compounds observed to show significant differences between closure technologies for the white wine. Figure 9 shows the MeSH trends for the four closures up to and including 24 months in bottle. Levels of MeSH have only increased under the screw cap closure across the twelve months since the previous testing point, with the wine showing a µg/l increase. similar effect has occurred with H 2S concentrations in the white wine since the previous testing at 12-months; small concentration increases are evident under all

17 Hydrogen Sulfide (µg/l) Methanethiol (µg/l) Commercial in Confidence closures, apart from the screw cap which has experienced a doubling in concentration from 1.2µg/L to 2.4 µg/l, on average. Results for the H 2S analysis in white wine can be seen in Figure 9. N.B. The shaded area represents the aroma threshold for methanethiol ( μg/l) Month 9 Month 12 Month 24 Month 2 0 Natural Cork Ref 1 Screw Cap (Saran/Tin) Vinolok 18.2 Vinolok 18.5 Figure 8: LMWS (Methanethiol) analysis up to 24-months post bottling in white wine samples N.B. The shaded area represents the aroma threshold for hydrogen sulfide ( μg/l) Month 9 Month 12 Month 24 Month Natural Cork Ref 1 Screw Cap Vinolok 18.2 Vinolok 18.5 (Saran/Tin) Figure 9: LMWS (Hydrogen Sulfide) analysis up to 24-months post bottling in white wine samples.

18 s seen with the red wine samples, DMS has shown significant development in the wine under all closures over the past 12-months. Whilst significantly above the sensory threshold (25µg/L), concentrations remain relatively low in comparison to the range of levels seen in commercial wines (0-980 µg/l). Within ppendix F there is a table summarising each of the detected low molecular weight sulfide compounds along with their odour descriptor, aroma detection threshold and typical range as found within ustralian commercial wines.

19 3.1.4 Sensory nalysis Of the 26 defined appearance, aroma and palate attributes, four attributes; yellow colour intensity, overall fruit aroma, floral aroma and sweaty/cheesy aroma, differed significantly (p<0.05) between closures for the white wine. Two attributes: dry grass aroma and hotness were close to significant (p<0.10). There were two attributes: sweet taste and astringency, that showed a significant replication effect, indicating that there was some variation between presentation replicates for these attributes. Table 4. Probability values and degrees of freedom (df) from the analysis of variance (NOV) for the white wine sample set. ttribute (C) Yellow Colour 0.007** Overall Fruit 0.032* Tropical Fruit Stonefruit Citrus Floral 0.025* Green Dry Grass 0.097ǂ Vegetal Box Hedge Cardboard Flint Sweaty/Cheesy 0.002** Pungent Overall Fruit F Tropical Fruit F Stonefruit F Citrus F Green F Sweet Viscosity cid Hotness 0.080ǂ stringency Bitter Fruit T df 3 Significance levels are as follows: * p < 0.05; ** p < 0.01; *** p < 0.001; ǂ p < df = degrees of freedom

20 Hotness P<0.1 stringency NTURL CORK SCREW CP VINOLOK 18.2 VINOLOK 18.5 Bitter Fruit T LSD = 0.28 Yellow Colour Intensity LSD = 0.44 Overall Fruit roma Tropical Fruit roma Stonefruit roma Citrus roma Viscosity Sweet cid LSD = 0.61 Floral Green roma Dry Grass P<0.1 Green Flavour Vegetal Citrus Flavour Box Hedge Stonefruit Flavour Tropical Fruit Flavour Overall Fruit Flavour Pungent Flint LSD = 0.76 Sweaty/Cheesy Cardboard Figure 10: Mean scores for all white wine attributes for the four different closures. LSD (5%) values included for significant attributes (p < 0.05). The wine bottled with the natural cork closure was significantly higher in yellow colour intensity and overall fruit aroma (Figure 10). Vinolok 18.2 had the second highest mean score for overall fruit aroma and was the highest in floral aroma while Vinolok 18.5 was intermediate in score for yellow colour intensity, overall fruit aroma and floral aroma. The wine bottled under screw cap was rated lowest in these characters, and highest in sweaty/cheesy.

21 Comparing the results to those gathered at the 12-month mark, there were a larger number of significant differences, although these tend to be due to differences between the screw cap and the other closures. Overall it appears as if the white wines under the Vinolok closures are displaying similar attributes to the wine under natural cork, with relatively high overall fruit and floral aromas. Of the 27 defined appearance, aroma and palate attributes for the red wine, only red fruit flavour and sweet taste showed a significant (p<0.05) closure effect - that is, there were statistically significant differences in the scores for these attributes between the different closures. There were also two attributes: floral aroma and hotness that were very close to significant (p<0.10). Table 5 shows the results of the NOV. Only vanilla aroma had a significant wine by judge interaction, indicating that the judges were consistent in scoring the wines in the same manner. Table 5. Probability values and degrees of freedom (df) from the NOV for the red wine sample set. ttribute Probability Opacity (Colour intensity) Overall Fruit Red Fruit Dark Fruit Confection Floral 0.098ǂ Green Cooked Vegetable Spice Vanilla Earthy Woody Pungent Overall Fruit F Red Fruit F 0.002** Dark Fruit F Green F Vanilla F Spice F Woody F Sweet 0.027* Viscosity cid Hotness 0.086ǂ stringency Bitter Fruit T df 3 Significance levels are as follows: * p < 0.05; ** p < 0.01; *** p < 0.001; ǂ p < 0.10.

22 NTURL CORK SCREW CP VINOLOK 18.2 VINOLOK 18.5 Bitter Fruit T Opacity Overall Fruit roma Red Fruit roma stringency 4.0 Dark Fruit roma Hotness Confection roma 2.5 cid 2.0 Floral 1.5 Viscosity Green roma Sweet LSD = Cooked Vegetable Woody Flavour Spice roma Spice Flavour Vanilla roma Vanilla Flavour Earthy Green Flavour Dark Fruit Flavour Red Fruit Flavour LSD=0.57 Pungent Overall Fruit Flavour Woody roma Figure 11: Mean ratings for red wine attributes for the four different closures. LSD values included for the attributes found to be significant across all samples The wine bottled under Vinolok 18.2 closure was rated significantly higher in red fruit flavour, followed by the wines bottled under screw cap and Vinolok 18.5, with natural cork rated the lowest. Vinolok 18.2 was also rated significantly higher for sweet taste than the other three closures. Natural cork was rated higher in floral aroma and Vinolok 18.5 was rated lowest in hotness, with these attributes close to significance (p<0.10) (Figure 11).

23 Compared to the results from the 12-month mark, there are still very slight differences for the red wine under the different closures. Overall, it appears as if the red wine under the Vinolok 18.2 closure is displaying similar attributes to the wine under the screw cap, with relatively high red fruit flavour. s was observed for the white wine sample set, there are relatively small differences between the closures at this time point.

24 OTR (cco 2 /day) Commercial in Confidence Wet OTR The results of the wet OTR analysis are shown in Figure 12. The graph compares data across three replicates of each of the four closure variants after the initial twenty-four months in bottle. The complete data set for each trial variant is provided in ppendix G Natural Cork - Ref 1 Screw Cap (Saran Tin) Vinolok 18.2 Vinolok Figure 12: Wet OTR analysis on the four closure variants at the 24-month time point There are significant differences in the measured closure OTR values. The screw cap (Saran/tin) had the lowest OTR value of ~0.002cc O 2 per day. The two Vinolok variants (18.2 and 18.5mm) were not significantly different with respect to OTR values. On average, the Vinolok 18.5mm closure had the highest OTR value of ~ cc O 2/day, compared to the Vinolok 18.2mm variant (~ cc O 2/day). There was a significant difference between both Vinolok closures and the natural cork (ref 1) closure. The increase observed for the natural cork samples since the 12-month time point highlights the closure repetition variability, however the range of values observed at the 24- month timepoint still lie within the indicative OTR values for this closure. s previously mentioned, testing conducted within Stage 1 (Fundamental Performance nalysis) indicated an OTR value of ~0.003cc O 2/day for the Vinolok 18.2 and 18.5mm closures, as measured on a dry basis. The current analysis conducted on samples stored for 24-months utilised a wet OTR method, whereby, on average, the Vinolok 18.2mm and Vinolok 18.5mm closures both indicated an OTR value of ~ cc O 2/day. Evidence presented within previous trials of a range of closure materials has shown that OTR values can increase over time, and this is likely attributable to the higher values presented above.

25 4 Summary Based on the results presented in this report, it can be summarised that: Significant, albeit small, differences exist in SO 2 levels at 24 months post bottling between wines under natural cork and all other closures types, consistent with those trends seen since the 12-month time point. There are no significant differences between wines under the Vinolok 18.2mm and 18.5mm closures. Minimal differences exist in wine colour parameters due to closure type after 24 months post bottling. There are no significant differences between wines under the Vinolok 18.2mm and 18.5mm closures for red wines; however small yet significant differences are present between these closures in white wine colour parameters OD320 and OD420nm at the 24-month time point. Differences in LMWS compounds were present in both red and white wine varieties at the 24-month time point. Carbon disulfide (CS 2) exhibited significant differences for the red wine under different closure types, with levels being lowest under the two Vinolok closures. Methanethiol (MeSH) and hydrogen sulfide (H 2S) exhibited significant differences for the white wine under different closure types, with levels under screwcap being highest The four closures showed statistical significance for yellow colour intensity, overall fruit aroma, floral aroma and sweaty/cheesy aroma in the white wine. Floral aroma was significantly higher in the white wine under Vinolok 18.2 sealed bottles than for other closure samples. The sweaty/cheesy attribute was highest under the screw-cap, which may be a reflection of higher concentrations of H 2S and MeSH that were present. Statistical significance was observed for red fruit flavour and sweet taste in the red wine. For both attributes, levels under the Vinolok 18.2 closure were higher than under the Vinolok 18.5mm closure. There was no significant difference in OTR value between the two Vinolok closure variants after twentyfour months in bottle, with the screw-cap (tin/saran) showing significantly lower OTR, as expected.

26 ppendices ppendix Basic Chemistry Table: Basic Chemistry of red wine samples bottled under (1) Natural Cork, (2) Screw Cap, (3) Vinolok 18.2 and (4) Vinolok 18.5 at 24 months. Titratable cidity Titratable cidity Volatile cidity as % lcohol ph ph 8.2 cetic cid Glucose/Fructose Specific Gravity verage StdDev verage StdDev verage StdDev verage StdDev verage StdDev verage StdDev verage StdDev Natural Cork Ref Screw Cap (Saran/Tin) Vinolok Vinolok Table: Basic Chemistry of white wine samples bottled under (1) Natural Cork, (2) Screw Cap, (3) Vinolok 18.2 and (4) Vinolok 18.5 at 24 months Titratable cidity Titratable cidity Volatile cidity as % lcohol ph ph 8.2 cetic cid Glucose/Fructose Specific Gravity verage StdDev verage StdDev verage StdDev verage StdDev verage StdDev verage StdDev verage StdDev Natural Cork Ref Screw Cap (Saran/Tin) Vinolok Vinolok Page 26 of 44

27 ppendix B SO 2 nalysis Results Table: White wine free SO 2 data up to 24 months post bottling Free SO2 (mg/l) 0 month 3 month 9 month 12 month 18 month 24 month verage StdDev verage StdDev verage StdDev verage StdDev verage StdDev verage StdDev Natural Cork Ref Screw Cap (Saran/Tin) Vinolok Vinolok Significance No - Yes - Yes - Yes - Yes - Yes - Table: Red wine free SO 2 data up to 24 months post bottling Free SO2 (mg/l) 0 month 3 month 9 month 12 month 18 month 24 month verage StdDev verage StdDev verage StdDev verage StdDev verage StdDev verage StdDev Natural Cork Ref Screw Cap (Saran/Tin) Vinolok Vinolok Significance No - No - Yes - Yes - Yes - Yes - Page 27 of 44

28 Table: White wine total SO 2 data up to 24 months post bottling Total SO2 (mg/l) 0 month 3 month 9 month 12 month 18 month 24 month verage StdDev verage StdDev verage StdDev verage StdDev verage StdDev verage StdDev Natural Cork Ref Screw Cap (Saran/Tin) Vinolok Vinolok Significance No - Yes - Yes - Yes - Yes - Yes - Table: Red wine total SO 2 data up to 24 months post bottling Total SO2 (mg/l) 0 month 3 month 9 month 12 month 18 month 24 month verage StdDev verage StdDev verage StdDev verage StdDev verage StdDev verage StdDev Natural Cork Ref Screw Cap (Saran/Tin) Vinolok Vinolok Significance No - Yes - Yes - Yes - Yes - Yes - Page 28 of 44

29 ppendix C Red Wine Colour nalysis Results Hue 3 month 9 month 12 month 18 month 24 month vg. SD vg. SD vg. SD vg. SD vg. SD Natural Cork Ref Screw Cap (Saran/Tin) Vinolok Vinolok Significance No - No - No - No - No - Colour Density 3 month 9 month 12 month 18 month 24 month vg. SD vg. SD vg. SD vg. SD vg. SD Natural Cork Ref Screw Cap (Saran/Tin) Vinolok Vinolok Significance Yes - No - No - No - No - Chemical ge 1 3 month 9 month 12 month 18 month 24 month vg. SD vg. SD vg. SD vg. SD vg. SD Natural Cork Ref Screw Cap (Saran/Tin) Vinolok Vinolok Significance No - No - No - No - No - Chemical ge 2 3 month 9 month 12 month 18 month 24 month vg. SD vg. SD vg. SD vg. SD vg. SD Natural Cork Ref Screw Cap (Saran/Tin) Vinolok Vinolok Significance No - No - No - No - No -

30 Free nthocyanins (mg/l) 3 month 9 month 12 month 18 month 24 month vg. SD vg. SD vg. SD vg. SD vg. SD Natural Cork Ref Screw Cap (Saran/Tin) Vinolok Vinolok Significance No - No - Yes - Yes - No - Total Phenolics 3 month 9 month 12 month 18 month 24 month vg. SD vg. SD vg. SD vg. SD vg. SD Natural Cork Ref Screw Cap (Saran/Tin) Vinolok Vinolok Significance No - No - No - No - No - Total Pigment 3 month 9 month 12 month 18 month 24 month vg. SD vg. SD vg. SD vg. SD vg. SD Natural Cork Ref Screw Cap (Saran/Tin) Vinolok Vinolok Significance No - No - Yes - Yes - Yes - Pigmented Tannin 3 month 9 month 12 month 18 month 24 month vg. SD vg. SD vg. SD vg. SD vg. SD Natural Cork Ref Screw Cap (Saran/Tin) Vinolok Vinolok Significance No - No - No - No - No -

31 ppendix D White Wine Colour nalysis Results Optical 280nm (a.u.) 0 month 3 month 9 month 12 month 18 month 24 month vg. SD vg. SD vg. SD vg. SD vg. SD vg. SD Natural Cork Ref Screw Cap (Saran/Tin) Vinolok Vinolok Significance Yes - Yes - Yes - Yes - Yes - Optical 320nm (a.u.) 0 month 3 month 9 month 12 month 18 month 24 month vg. SD vg. SD vg. SD vg. SD vg. SD vg. SD Natural Cork Ref Screw Cap (Saran/Tin) Vinolok Vinolok Significance Yes - Yes - No - Yes - Yes - Optical 420nm (a.u.) 0 month 3 month 9 month 12 month 18 month 24 month vg. SD vg. SD vg. SD vg. SD vg. SD vg. SD Natural Cork Ref Screw Cap (Saran/Tin) Vinolok Vinolok Significance Yes - Yes - Yes - Yes - Yes -

32 ppendix E Low Molecular Weight Sulfide results: Red Wine Carbon disulfide (μg/l) 0 month 9 month 12 month 24 month vg. SD vg. SD vg. SD vg. SD Natural Cork Ref Screw Cap (Saran/Tin) Vinolok Vinolok Significance No - Yes - Yes - Yes - Dimethyl sulfide (μg/l) 0 month 9 month 12 month 24 month vg. SD vg. SD vg. SD vg. SD Natural Cork Ref Screw Cap (Saran/Tin) Vinolok Vinolok Significance No - Yes - Yes - No - Hydrogen Sulfide (μg/l) 0 month 9 month 12 month 24 month vg. SD vg. SD vg. SD vg. SD Natural Cork Ref Screw Cap (Saran/Tin) Vinolok Vinolok Significance No - Yes - Yes - No - Methanethiol (μg/l) 0 month 9 month 12 month 24 month vg. SD vg. SD vg. SD vg. SD Natural Cork Ref Screw Cap (Saran/Tin) Vinolok Vinolok Significance No - Yes - Yes - No -

33 Methyl Thioacetate (μg/l) 0 month 9 month 12 month 24 month vg. SD vg. SD vg. SD vg. SD Natural Cork Ref Screw Cap (Saran/Tin) Vinolok Vinolok Significance No - Yes - Yes - No -

34 ppendix F Low Molecular Weight Sulfide results: White Wine Carbon disulfide (μg/l) 0 month 9 months 12 months 24 month vg. SD vg. SD vg. SD vg. SD Natural Cork Ref 1 < Screw Cap (Saran/Tin) < Vinolok 18.2 < Vinolok 18.5 < Significance No - Yes - Yes - No - Dimethyl sulfide (μg/l) 0 month 9 months 12 months 24 months vg. SD vg. SD vg. SD vg. SD Natural Cork Ref Screw Cap (Saran/Tin) Vinolok Vinolok Significance No - Yes - Yes - No - Hydrogen Sulfide (μg/l) 0 month 9 month 12 months 24 months vg. SD vg. SD vg. SD vg. SD Natural Cork Ref 1 < < Screw Cap (Saran/Tin) < < Vinolok 18.2 < < Vinolok 18.5 < < Significance No - Yes - Yes - Yes - Methanethiol (μg/l) 0 month 9 month 12 month 24 month vg. SD vg. SD vg. SD vg. SD Natural Cork Ref Screw Cap (Saran/Tin) Vinolok Vinolok Significance No - Yes - Yes - Yes -

35 Low molecular weight sulfide compounds and their respective sensory thresholds. Compound Odour Descriptor roma Threshold (µg/l) Typical range (µg/l) Hydrogen sulfide Rotten egg, sewage like Methanethiol Rotten cabbage, burnt rubber, putrid Dimethyl sulfide Carbon disulfide Blackcurrant, cooked cabbage, asparagus, canned corn, molasses Sweet, ethereal, slight green, rubber, sulfidy, chokingly repulsive > Methyl thioacetate Sulfurous, cheesy, egg

36 ppendix G Oxygen transmission rate results verage StDev Vinolok Vinolok Natural Cork - Ref Screw Cap (Saran Tin)

37 ppendix H Sensory nalysis (White Wine Samples) Natural Cork Sample Yellow Colour I Overall Fruit Tropical Fruit Stonefruit Citrus Floral Green Dry Grass Vegetal Box Hedge Cardboard Flint Sweaty/Cheesy Rep Rep Rep Mean Standard deviation Pungent Natural Cork Sample Overall Fruit F Tropical Fruit F Stonefruit F Citrus F Green F Sweet Viscosity cid Hotness stringency Bitter Fruit T Rep Rep Rep Mean Standard deviation Page 37 of 44

38 Screw Cap Sample Yellow Colour I Overall Fruit Tropical Fruit Stonefruit Citrus Floral Green Dry Grass Vegetal Box Hedge Cardboard Flint Sweaty/Cheesy Rep Rep Rep Mean Standard deviation Screw Cap Sample Overall Fruit F Tropical Fruit F Stonefruit F Citrus F Green F Sweet Viscosity cid Hotness stringency Bitter Fruit T Rep Rep Rep Mean Standard deviation Pungent Page 38 of 44

39 Vinolok 18.2 Sample Yellow Colour I Overall Fruit Tropical Fruit Stonefruit Citrus Floral Green Dry Grass Vegetal Box Hedge Cardboard Flint Sweaty/Cheesy Rep Rep Rep Mean Standard deviation Vinolok 18.2 Sample Overall Fruit F Tropical Fruit F Stonefruit F Citrus F Green F Sweet Viscosity cid Hotness stringency Bitter Fruit T Rep Rep Rep Mean Standard deviation Pungent Page 39 of 44