Methods and compositions for fining fermentable beverages

Abstract

The present disclosure relates to methods and compositions for fining fermentable beverages, such as alcoholic fermentable beverages, for example, wine, beer, lager, ale, cider, perry, spirit and liqueur. The compositions herein comprise a pectin and carrageenan which are used to treat the beverage prior to fermentation.

Claims

1. A method for fining fermentable beverages comprising treating the beverage with a fining composition consisting of: (i) a low methyl ester or low methoxy (LM) pectin having a degree of esterification (DE) of 50% or less; (ii) a carrageenan; and (iii) 0.1 to 2 g/L of bentonite, wherein the fining composition is added to the fermentable beverage prior to fermentation.

2. The method according to claim 1, wherein the low methyl ester or low methoxy (LM) pectin has a DE of 0-35%.

3. The method according to claim 2, wherein the LM pectin is a low methyl ester (LMC) pectin or an amidated, low methyl ester (LMA) pectin.

4. The method according to claim 1, wherein the pectin is standardised pectin or an active pectin.

5. The method according to claim 3, wherein the pectin is in the form of an alkali metal salt.

6. The method according to claim 5, wherein the alkali metal salt is a sodium or potassium salt.

7. The method according to claim 1, wherein the amount of pectin added when the beverage is an acidic beverage having a calcium level of 70 mg/l or above is 0.2-5 g/L.

8. The method according to claim 1, wherein the carrageenan is a cold water soluble type or a cold water insoluble type.

9. The method according to claim 8, wherein the cold water soluble type carrageenan is an iota-dominated carrageenan and the cold water insoluble type carrageenan is a kappa-dominated carrageenan.

10. The method according to claim 1, wherein the amount of carrageenan added to the fermentable beverage is 0.02 to 0.2 g/L in liquid form or 0.05 to 0.5 g/L in powder form.

11. The method according to claim 1, wherein the fermentable beverage is a fruit juice or fruit which is subsequently crushed and wherein the pectin and carrageenan are either removed from the juice or fruit during racking or remain in the juice or fruit during fermentation.

12. The method according to claim 1, wherein the fermented beverage is a fermentable alcoholic beverage.

13. The method according to claim 1, wherein the bentonite is present in an amount of 0.1 to 1 g/L.

Description

DESCRIPTION OF THE DRAWINGS

(1) In the Examples which follow, reference will be made to the accompanying drawings in which:

(2) FIG. 1 is a graph showing the effect of juice treatment with bentonite, pectin and carrageenan on alcohol formation during wine fermentation. Fermentation was controlled at 16° C. for 12 days. Control was the fermentation without any addition. Bentonite was used as a positive control with dosage rate of 1 g/L. CB-L038/06 was dosed as powder form at 1.5 g/L in the grape juice and CSW-2 was dosed as 2% solution to 0.2 g/L in the juice in either individual dosage or the combination.

(3) FIG. 2 is a graph showing wine calcium reduction during wine fermentation. Calcium concentration was analysed at day 0, day 6 and day 12 and compared to the control. The dosage rate and the method for addition of each additive are described in FIG. 1 above.

(4) FIG. 3 is a graph showing changes in wine volatiles after 12 day fermentations of untreated, and bentonite- or carrageenan+pectin-treated juice. Figures A, B and C show esters, higher alcohols and acids formation. The dosage rates and the method for addition of each additive is described in FIG. 1 above.

(5) FIG. 4 is a graph showing changes in wine stability after 12 day fermentation using the treated juice.

EXAMPLES

(6) The present invention will now be described with reference to the following non-limiting examples.

(7) In the examples, reference will be made to the following abbreviations:

(8) TABLE-US-00001 Ethyl acetate ETOAC Ethyl hexanoate ETHEX Ethyl octanoate ETOCT Phenyl ethyl acetate PHETAC Isoamyl acetate IAMOAC Total esters TOTEST n-propanol NPROH Isobutanol IBUOH Isoamyl alcohol IAMOH Phenyl ethyl alcohol PHETOH Total alcohol TOTALC Butanoic acid BUT Hexanoic acid HEX Octoanoic acid OCT Decanoic acid DEC Total acid TOTACD
Materials and Methods:
Raw Materials

(9) The white chardonnay juices used in the examples were freshly supplied by Great Western Wineries, Victoria. The juices were either stored frozen or used immediately.

(10) Dry wine yeast QA23 (Danstar Ferment AG, Switzerland) was used for the fermentation trials. The yeast was hydrated by addition of 10 volumes of distilled water at 35-40° C. for 20 min without shaking. Thereafter the slurry was mixed well prior to pitching.

(11) A water-soluble type carrageenan (CSW-2) was supplied by CP Kelco. Copenhagen, Denmark. 2% CSW-2 solution was freshly prepared following the standard preparation protocol. The pectins CU-L024/05, CB-L038/06 and CU-L023/05, were supplied by Herbstreith & Fox Gmb, Nurnberg, Germany. They are non-amidated LMC citrus pectins. The DE of each pectin is 25%, 10% and 7% respectively. The pectin, TS1580 was supplied by CP Kelco (DE ˜18%, trial lot).

(12) Samples of juice, treated and untreated fermented juice and the final wine products (post-filtration) were collected and calcium concentration, alcohol, pH and volatiles were measured.

(13) Estimation of Calcium and Other Metal Ions in Wine

(14) Metal ions are measured by directly injecting 5 μl of wine sample into a HPLC (HP 1100) system equipped with a conductivity detector (Waters 432). A Universal cation column (100×4.6 mm×7μ) was used for separation.

(15) Method for Wine Volatiles Analysis (GC-Headspace)

(16) A gas chromatograph equipped with temperature programing, mass spectrometer, capillary/splitless injector port system, headspace auto-sampler and BP21 capillary column (25 m×0.32 mm×0.25μ) was used for the analyses. Approximately 2 g sodium chloride was added to headspace vials (10 ml); 3 mL of wine sample was injected into the headspace vials. The sample vial was immediately crimped shut, 10 μL of internal standard was injected. The volatiles were estimated by the CC/MS by comparison to the internal standard.

(17) Method for Wine Heat-Stability Test

(18) About 30 ml wine sample was filtered through a 0.45 μm syringe filter (Sartorius). The filtered sample was heated at 80° C. for 2 h and cooled down to 4° C. for another 2 h prior to turbidity measurement.

(19) The turbidity of the heated samples was measured using a Hach turbidimeter (2100 AN) and is quoted as nephelometry turbidity units (NTU). The difference in the turbidity before and after heating should be, ideally, less than or equal to 2 NTU for stable wine.

Example 1—Suitable Carrageenans and Pectins for Wine Stabilisation

(20) Carrageenan

(21) Different types of carrageenan can have different water solubilities and also different protein binding capacities and affinities. The suitability of different carrageenans to treat white grape juice was assessed using the wine heat stability test. Fermentations were carried out in bench-top scale (500 ml) fermenters.

(22) CSW-2, an iota-dominated carrageenan was dissolved in distilled water to 2% w/v at room temperature. K-100, a defined kappa-dominated carrageenan was hydrated in water at 60° C. to 2% w/v solution.

(23) A LMC pectin, CB-L038/06 was also included in this example.

(24) The carrageenan or pectin solution was mixed with the grape juice at room temperature (22° C. 200 mg/L) and stirred for 15 min.

(25) The juice was transferred into a cylinder and allowed to settle at room condition for 16 h. The treated juice was then transferred in 1 L Schott bottles and freshly prepared yeast slurry was subsequently added to 200 mg dry weight yeast/L. The juice was allowed to ferment at 22° C. in the bottles for 10 days.

(26) TABLE-US-00002 TABLE 1 Treatment of white grape juice with carrageenan and pectin. Fermentations were carried out in 1 L Schott bottles at 22° C. Ethanol concentration and wine stability were measured after 10 day fermentations. CSW-2 is cold water soluble carrageenan, K-100 is a commercial kappa- carrageenan and CB-L038/06 is a LMC pectin. CSW-2 K-100 CB-L038/06 Control (0.2 g/L) (0.2 g/L) (0.2 g/L) Ethanol (%, v/v) 12.2 12.6 12.4 12.7 Heat-turbidity (NTU) 49.0 4.6 8.9 31.4

(27) Results after fermentation in Table 1 show that final ethanol production was not affected by carrageenan or pectin. In fact, fermentations with the treated juice were observed to be slightly faster than the control as well reaching slightly higher terminal ethanol concentration. The wine made from the pectin-treated juice was slightly more stable than control (NTU 31.4 versus 49.0). But the heat-stability test result was still well over the limit specified for packaged white wine product (2 NTU) However, the wines made with either of the carrageenan-treated juices were much more stable than either the control or the wine after pectin treatment. Carrageenan, CSW-2 treatment was the most effective. The turbidity of the (CSW-2) wine was only 4.6, the (K-100) wine was 8.9, ˜90% and ˜80% reduction respectively, over the control. CSW-2 (cold-water soluble type) was more effective than K-100 (cold water insoluble type) as K-100 will form a gel very quickly in cold grape juice (<10° C., pH˜3.2). CSW-2 is a cold water soluble carrageenan and therefore will have a much longer contact time with juice proteins before gelling out of the juice.

(28) Pectin

(29) We previously found that some calcium-reactive LMC pectins, such as CU-L024/05 (DE=25%) will remove significant amounts of calcium from white wine when delivered in powder form. We were keen to determine whether this would happen in grape juice. Wine is different to grape juice because of the 12-14% ethanol content, but commercial grape juice is ‘ethanol-free’.

(30) Bench top fermenters were used to investigate the effects on fermentation and stability of combinatorial dosing of pectins and carrageenans into white grape juice. In the first instance, the ability of pectins to remove calcium from grape juice was tested.

(31) Four LMC, calcium-reactive pectins were investigated. These pectins were chosen after a broaden review of pectin types, based on DE levels and calcium reactivity. Powder pectin was thoroughly dispersed into grape juice at 1 g/L at 10° C., using a magnetic stirrer to agitate for 15 min. The juice was allowed to settle at 4° C. for at least 16 h before samples were taken for calcium analysis. The results are shown in Table 2. Each pectin (1 g/L) was able to reduce juice calcium. CU-L024/05 was the least effective—at 15% reduction; CB-L038/06 was the best as it removed almost 23%.

(32) These results show that pectins with DE levels ranging between 10-18% give the best calcium reduction rate in grape juice.

(33) TABLE-US-00003 TABLE 2 The effect of pectin addition on calcium reduction from grape juice. Pectin was added to 1.0 g/L in the juice at 10° C. with agitation. ΔCa (%) indicates a percentage of calcium being removed from the juice. CU- CB- CU- control L024/05 TS1580 L038/06 L023/05 Pectin DE (%) — 25 18 10 7 Ca.sup.2+ (mg/L) 89 75 70 69 72 ΔCa (%) — −15.7 −21.3 −22.5 −19.1

Example 2—Effect of CSW-2 and CB-L038/06 Additions on Fermentation Rate and Alcohol Production

(34) CSW-2 and CB-L038/06 were selected for further fermentation trials (20 L level). Five fermenters were used. 2007 vintage chardonnay juice was treated with either CSW-2 (0.2 g/L), CB-L038/06 (1.5 g/L) or a combination of both. Untreated juice was the control. Bentonite (1 g/L) addition served as a positive control. Each juice was transferred into the fermenter immediately after treatment. It was chilled to 10° C. for 16 h before the hydrated wine yeast—QA23 was inoculated. Fermentation was controlled at 16° C. for 32 days.

(35) FIG. 1 shows that the treated grape juice fermented faster than the control. Alcohol production after fermentation of treated juice was practically the same at 11.8% (v/v). Alcohol in the untreated juice was only 10.3% after 12 days. After 16 days it had almost reached 11.5% (not shown).

Example 3—Effect of CSW-2 and CB-L036/06 Grape Juice Treatment on Calcium, Volatiles and Wine Stability

(36) Calcium concentration during fermentation of treated grape juice were monitored at days 0, 6 and 12.

(37) The calcium concentration in the untreated juice was >115 mg/L at the start of fermentation (FIG. 2). Addition of either bentonite or CSW-2 (carrageenan) did not reduce juice calcium. However, the addition of CB-L38/06 (alone or in combination with carrageenan) resulted in a significant drop in juice calcium. An even more obvious calcium reduction occurred in the finished wine which showed a 28.4% reduction with pectin alone and 32.4% in combination with carrageenan. This is presumably because pectin gelation/precipitation in wine is more effective because of the alcohol.

(38) Bentonite and carrageenan both reduced calcium by ˜10% after fermentation, which is still significant.

(39) Wine volatiles (esters, higher alcohols and acids) were monitored during fermentation. The esters (FIG. 3A) in the CSW-2 and CB-L038/06 wines increased by >21% (ethyl hexanoate) up to 140% (phenylethyl acetate). This adds up to a 49% increase in total ester production. In bentonite treated juice, ethyl hexanoate production dropped by 11% but ethyl octanote production increased by 71%. Overall there was no change in total ester production with bentonite-treated grape juice.

(40) Carrageenan and pectin co-treatment led to an increase in most higher alcohols (FIG. 3B) except n-propanol, which dropped slightly by 7%. Total higher alcohol production was up by 34%, compared to ˜14% for bentonite treated juice. Again the carrageenan-pectin co-treatment resulted in a 110% increase in octanoic acid, but a 33% drop in decanoic acid level. Overall total acids increased by 27%. In bentonite-wine total acids were down by 10%.

(41) The wines were also evaluated by a house sensory panel. 8 out of 9 testers preferred the wine that was made from the carrageenan-pectin co-treated juice. It was considered fruiter and truer to ‘chardonnay-type’.

(42) In contrast, although the chemical data indicated that bentonite treatment did not markedly affect wine volatiles, the panel thought this wine was thin and a bit ‘earthy’. Bentonite did strip out colour compared to the control whereas the other treatments did not (Table 3).

(43) TABLE-US-00004 TABLE 3 Changes in wine pH and colour caused by the juice treatments. CSW-2 + control Bentonite CB-L38/06 pH 3.6 3.5 3.6 Colour (EBC unit) 2.4 1.5 2.3
Heat Stability

(44) Wines were chilled to 4° C. and incubated for 5 days, filtered through a Millipore glass fibre filter and then assessed for heat-stability. The control wine was ˜60 NTU (FIG. 4), CB-L038/06-wine gave a value of 40 NTU. This is far from a ‘significant’ improvement. Carrageenan (CSW-2) treatment of juice produced a very low NTU result in the wine—down to 5.2, with pectin+carrageenan this dropped to 4.7. Wine makers in Australia typically aim for a value of 2 or less. Bentonite (1.0 g/L)-wine tested at 1.4 NTU, is well below this value.

(45) This example shows that grape juice treatment may be a more convenient, simpler way to stabilize wine than current practice, which treats wine after the fermentation is complete. Bentonite by itself has a very limited ability to remove calcium from wine, when added either pre- or post-fermentation. Pectin stands out as a very effective agent for calcium removal.

(46) In combination with carrageenan it appears to be slightly more effective. Carrageenan removes protein yet has almost no negative effects on volatile levels. Bentonite is even Letter at protein removal, thus carrageenan in combination with a very low dose of bentonite ought to achieve <2 NTU in the heat test without any, or little effect on wine volatiles. A combination of bentonite, carrageenan and pectin may be the best ‘recipe’ for wine stabilization. It also provides plenty of scope for wine maker versatility.

Example 4—Effect of Bentonite Addition During Grape Juice Treatment on Wine Stability

(47) Past practice has invariably been about treatment of wine with soluble macromolecules. This requires the use of dosing pumps and associated equipment that adds cost and complexity to wine making and limits the application to medium or large wineries. Even then it may not be considered financially justified. Therefore as well as assessing soluble process aids, we have looked for macromolecules that are equally effective when introduced as the powder. This creates far more versatile products and more simple ease of application.

(48) Such a combination for the treatment of grape juice is described in the following section. Bentonite (0.4 g/L) was combined with carrageenan CSW-2 (0.1 g/L) and pectin TS1580 (1.0 g/L). The powder mixture was used to treat grape juice that was subsequently used for laboratory bench-top fermentations. Untreated juice and a mixture without bentonite (CSW-2+TS1580) were used as controls. The mixtures were added to the juices thoroughly in 1 L Schott bottles for 15 min before allowing the samples to settle at 4° for at least 16 h. 0.2 g/L dry wine yeast QA23 was then hydrated in distilled water and inoculated into each bottle. Fermentation was then allowed to proceed at room temperature (22° C.) for 10 days.

(49) The results (Table 4) show that volatile formation in treated juices increased significantly compared to the control, as expected. Final pH, alcohol level and wine colour were not changed however. Wine calcium concentration in the treated wines was about 25% lower compared to the control. Sodium concentration in both cases was up from 12 mg/L to 84-85 mg/L. This is still within the acceptable range for Australian white wine. Importantly, both treatments produced more stable wines. The control wine scored 32 NTU in the heat test. The (pectin+carrageenan)-wine scored 5.8 NTU. The wine made from grape juice treated with pectin, carrageenan and bentonite was 2.7 NTU. This indicates that a one-step process would be feasible to replace the post fermentation use of a high dose of bentonite for heat stabilisation. Bentonite+CSW-2+TS1580 treatment, is one such option.

(50) TABLE-US-00005 TABLE 4 Effects of grape juice treatment on wine volatiles formation, chemicals and physical properties. Diff Diff 1 2 (+/− %) 3 (+/− %) Esters Ethyl acetate 28.00 39.80 42.1 41.20 47.1 Ethyl hexancate 0.42 0.92 119.0 0.96 126.6 Ethyl actanote 0.44 0.72 63.6 0.76 72.7 Phenylethyl 0.28 0.60 114.3 0.62 121.4 acetate Isoamyl acetate 0.96 1.96 104.2 2.18 127.1 higher n-Propanol 53.60 53.20 −0.7 52.80 −1.5 Isobutanol 31.80 43.20 35.8 42.20 32.7 Isoamyl alcohol 118.60 125.80 6.1 126.00 6.2 Phenylethyl 38.00 35.00 −7.9 35.00 −7.9 alcohol fatty Hexanoic acid 2.50 5.08 103.2 5.56 122.4 acids Octanoic acid 4.28 7.08 65.4 7.56 76.6 Decanoic acid 1.14 1.62 42.1 1.80 57.9 alcohol (v/v, %) 10.43 10.71 2.7 10.73 2.9 pH 3.42 3.42 0.0 3.42 0.0 Colour 1.60 1.70 6.3 1.70 6.3 Ions Ca.sup.2+ 98.00 74.00 −24.5 72.00 −26.5 Na.sup.+ 12.00 85.00 608.3 84.0 600.0 Heated Haze 32.00 5.8 −82.0 2.7 −91.5 1: untreated control, 2: CSW-24 + TS1580 and 3: bentonite + CSW-2 + TS1580. Diff. (+/− %) indicates changes between the test and the control. Increase by 10% or more is highlighted in bold while decrease by 10% or more is hiebliahted in italics.

CONCLUSION

(51) Fermented beverages such as wine have exceptional complexity. Post-fermentation treatments to achieve heat and cold stabilization generally do influence wine flavour characteristics.

(52) The stabilization of fermentable beverages such as grape juice in the case of wine is a different way of approaching beverage stabilisation. The point of adding pectin into grape juice, may seem counterintuitive, when treatment with pectinase is frequently used to avoid filtration problems downstream after fermentation. Macromolecular processing aids, are generally added as solutions or colloidal suspensions, which can be problematic, as this requires dosing and delivery equipment that adds to overheads, and limits the uptake of new technology. Dedicated systems usually lead to some loss of plant flexibility.

(53) The method and composition of the present invention reduce some of these problems. Carrageenan and pectin are ‘generally regarded as safe (GRAS)’ ingredients. Pectin occurs naturally in all plants, and although differences in structural complexity occur, the general structure of this polygalacturonic acid is the same. Carrageenan is also a food processing aid that is recovered in commercial quantities from marine plants. Powder mixtures of pectin and carrageenan can be dosed by manual addition, or by auger, or other mechanical delivery systems.

(54) There are potential benefits for using these beverage pre-treatments. It has been found that the fermentations are faster, the final ferments are clearer, while there appears no change of volatile profile, no loss of complexity using these treatments. Pectin, carrageenan and bentonite are traditional food ingredients. The stoichiometry of the mixture can be varied readily depending on the molecular “targets”, and/or cations to be removed. The addition of these macromolecules does not detract from the art of the wine maker, from his or her implicit skills, and mystery of the process.

(55) Climate change effects are predicted to increase in the short term. Calcium accumulation in fruit is just one of the consequences of climate changes, probably brought on as plants optimize osmotic gradients for water accumulation. There is also likely to be a change in the residual protein level in grape juice and possibly an increase in the recalcitrant character of some of these proteins. Effective removal of heat-sensitive protein and the optimization of premium wine capacity from all vintages will be very important as fruit yields decline as hydrotherms move north and south, and affect the traditional vine growing districts.

(56) The method and composition of the present invention will promote fruiter wines, more stable wine, and eliminate, at least largely diminish the waste production in wine making, which again is a critical profitability issue for marginal producers.

(57) Addition of a mixture of pectin and carrageenan into a fermentable beverage such as grape juice removes the majority of unstable proteins and a significant amount of calcium. Wine calcium can be controlled by dosing appropriate levels of pectin in grape juice. Wines made from the treated juice are fruiter with a fuller body. Wine stability is also significantly improved by the treatment. Therefore wine may only require a ‘polishing’ process or even no further stabilization treatment when treated by the method of the present invention.

Example 4—Vintage Trials with Chardonnay and Sauvignon Blanc

(58) Vintage trials were carried out with Chardonnay and also Sauvignon Blanc juice to evaluate the effect of pectin and carrageenan treatment on bentonite requirement. In addition the effects of these treatments on wine volatiles were compared, together with the fermentation rates for the treated and untreated juice. In this example, the calcium levels were below the vintage maximum threshold for calcium, and consequently the treatments were not designed to reduce the final calcium levels after fermentation by a significant margin.

(59) The following trials used Chardonnay grapes grown in the Victorian Central highland district and Sauvignon Blanc grapes also grown in this region.

(60) Vintage 2008

(61) Trial 1

(62) Grapes were harvested, and treated according to the conditions described below. In Part A, the carrageenan solution and pectin powder were added to the Chardonnay grape juices, with mild agitation of the demijohn containers. In Part B, pectin powder (TS 1580) and carrageenan (CSW-2) solution were added directly to the grapes (45 kg), mixed manually to get an even distribution, and subsequently crushed using a small, hand-operated press.

(63) In Part A the flowing treatments were set up: Control A—no pectin or carrageenan addition. Treatment 1A—Addition of 1.5 g/L pectin (powder form) and 0.150 g/L carrageenan (liquid) and incubation for 30 min before the addition of pectinase and chilling (duplicate). Treatment 2 A—Addition of 1.5 g/L pectin and 0.150 g/L carrageenan and incubation for 1 hour before pectinase addition and chilling (duplicate). Treatment 3A—Addition of 1.5 g/L pectin and 0.150 g/L carrageenan and incubation for 2 hours before pectinase addition and chilling (duplicate).
Samples for yeast available nitrogen (YAN) analysis were collected prior to pectinase addition and analysed by digestion of the sample with arginase and urease to liberate ammonia. The ammonia is quantitated using an enzymation protocol in which NADPH coenzyme formation is measured Spectrophotometrically during conversion of 2-oxoglutarate to L-glutamate in the presence of glutamate dehydrogenase.

(64) In Part B the following tests were set up: Control B—no pectin or carrageenan addition. Treatment 1B—Addition of 1.5 g/L pectin and 0.150 g/L carrageenan to 45 kg of fruit followed by the usual mix and crush and processing. Treatment 2B—Addition of 1.5 g/L pectin and 0.150 g/L carrageenan to 45 kg of fruit and hand-mix in, then incubation for 1 hour before processing as per normal. Treatment 3B—Addition of 1.5 g/L pectin and 0.150 g/L carrageenan to 45 kg of fruit and mix in then incubation for 2 hours, prior to processing as per normal.

(65) Following incubation, 30 ppm (0.4 ml) pectinase was added and the demijohns were placed at 4° C. for settling for 18 hours before decanting.

(66) EC1118 yeast (300 mg/L) was inoculated into the juice once the demijohns were warmed to 16° C.

(67) Fermentation was between 18-20° C. and the fermentation rate was monitored by measuring Bé. The final Bé was 0. Once each ferment was finished, it was placed at 4° C., 60 mg/L SO.sub.2 was added and it was allowed to settle. The wine was racked and bottled under screw caps for general quality analysis, including metal ions, volatiles and heat stability.

(68) TABLE-US-00006 TABLE 3 Treatment data for Trial 1, Part A and Part B. The Chardonnay juices were treated by a combination of carrageenan solution and pectin powder and settled at 4° C. for 16 h. Wine yeast EC1118 was inoculated into the juice and then fermented at ambient (20° C.) temperature for 7 days. Volume 10% Juice after EC1118 Demijohn treated racking added Additional Trial code (DJ) No. (L) (L) (ml) information Part A Chardonnay grape juice Control 1A D13 12 9.5 30 Bé 10.8, Control 2A D14 12 9.3 30 pH 3.37, TA 7.2, YAN 217 FSO.sub.2 8 mg/l T 1A D9 12 9.6 30 D10 12 9.25 30 T 2A D11 12 8.8 30 D16 12 8.4 30 T 3A D17 12 8.6 30 YAN 206 D18 12 8.25 30 Part B Grape - crash - juice collected Control B D29 12 10.0 30 T 1B D30 12 8.5 30 T 2B D27 12 9.0 30 T 3B D28 12 8.85 30

(69) TABLE-US-00007 TABLE 4 Fermentation data for Trial 1, Part A and Part B Day 1 Day 4 Day 7 DJ No. Bé Temp Bé Temp Bé Temp Juice Control A 13 10.8 16 4.8 23 0 20 14 10.8 16 4.4 23 0 20 1A 9 10.8 16 4.6 23 0 20 10 10.8 16 4.6 23 0.2 20 2A 16 10.8 16 4 23 0 20 11 10.8 16 4.6 23 0.4 20 3A 17 10.8 16 4.2 23 0.2 20 18 10.8 16 4.2 23 0 20 Fruit Control B 29 11 16 5.6 23 0.4 20 1B 30 11 16 5.4 23 0.2 20 2B 2 11 16 4.8 23 0 20 3B 28 11 16 5.8 23 0.2 20

(70) Table 4 records the changes in sugar levels (Bé) during the 7 day fermentation for Part A (addition to grape juice). After Day 4, the Bé values for the control and for T1A, 2A and 3A (Chardonnay, pectin and carrageenan, 0.5, 1 and 2 h incubation) were very similar. And after 7 days it was concluded that the differences in residual sugar were not significant. Similarly the fermentation rate for Part B (pectin and carrageenan to grapes) showed no major difference at Day 4 and Day 7.

(71) TABLE-US-00008 TABLE 5 Volatile analysis for wine made in Trial 1, Part A and Part B Control Control A 1A 2A 3A B 1B 2B 3B ETOAC 38.1 43.1 41.0 41.0 38.4 40 41.6 45.7 ETHEX 1.0 1.2 1.2 1.1 1.16 1.12 1.08 1.14 ETOCT 1.0 1.2 1.1 1.0 1.1 1.05 0.84 1.06 PHETAC 0.3 0.4 0.3 0.3 0.38 0.41 0.38 0.41 IAMOAC 3.1 3.7 3.2 3.0 3.06 3.14 3.26 3.89 TOTEST 43.5 49.6 46.8 46.4 44.1 45.7 47.2 52.2 NPROH 44.0 43.4 42.1 40.7 37.6 40.5 37.5 45.0 IBUOH 19.5 19.3 19.7 19.5 18.2 19.0 19.7 19.6 IAMOH 132.8 122.4 122.4 123.8 129.2 120.4 132 120.8 PHETOH 28.1 23.4 24.0 24.1 29.6 28.1 29.4 24.1 TOTALC 224.4 208.2 208.1 208.0 214.6 208 218.6 209.7 BUT 1.5 1.7 1.6 1.6 1.5 1.4 1.5 1.4 HEX 4.9 5.6 5.4 5.0 5.0 5.1 4.5 5.0 OCT 9.0 10.6 9.9 9.3 9.7 9.2 8.5 9.0 DEC 3.1 3.8 3.6 3.2 3.4 3.4 2.7 3.4 TOTACD 18.8 21.9 20.7 19.4 19.9 19.6 17.5 19.1 Alc (%) 11.1 11.1 11.1 11.0 11.2 11.2 11.0 11.4

(72) TABLE-US-00009 TABLE 6 The percentage changes in volatiles for Trial 1, Part A and Part B (cf. Table 7) Control Control % A 1A 2A 3A B 1B 2B 3B ETOAC 0 13.3 7.8 7.6 0.0 4.2 8.3 19.0 ETHEX 0 19.9 14.1 7.3 0.0 −3.4 −6.9 −1.7 ETOCT 0 14.3 10.8 3.0 0.0 −4.5 −23.6 −3.6 PHETAC 0 12.3 0.0 −6.2 0.0 7.9 0.0 7.9 IAMOAC 0 19.4 4.1 −2.9 0.0 2.6 6.5 27.1 TOTEST 0 13.9 7.6 6.7 0.0 3.6 7.0 18.4 NPROH 0 −1.4 −4.3 −7.6 0.0 7.7 −0.3 19.7 IBUOH 0 −1.0 0.8 −0.3 0.0 4.4 8.2 8.8 IAMOH 0 −8.0 −7.9 −6.8 0.0 −6.8 2.2 −6.5 PHETOH 0 −16.9 −14.8 −14.4 0.0 −5.1 −0.7 −18.6 TOTALC 0 −7.2 −7.3 7.3 0.0 −3.1 1.9 −2.3 BUT 0 12.2 5.8 7.5 0.0 −1.4 0.7 −5.4 HEX 0 14.0 10.2 2.8 0.0 2.6 −9.5 1.6 OCT 0 17.9 10.2 3.7 0.0 −4.4 −12.0 −7.3 DEC 0 23.9 15.5 3.9 0.0 0.0 −20.9 0.6 TOTACD 0 16.6 10.1 3.3 0.0 −1.8 −11.7 3.8

(73) The wine volatile data for Part A and B wines showed variations in some volatiles. The total esters in absolute figures do not show major differences. Pre-treatment with pectin and carrageenan of grape juice (1A, 2A and 3A) increased the total esters by about 10%. Likewise the pre-treatment of grapes elevated total esters by about the same amount. Higher alcohols decreased slightly. Total acids increased slightly. If the percentage changes are compared (Table 6), the changes are as follows: pre-treatment of grape juice (Part. A) enhances total esters up to 14%; pre-treatment of grapes (Part B) increases esters by up to 18%. Total higher alcohols (Part A vs B, Table 8) dropped by slightly over 7% and 3% respectively. Total acids were up, 16.6% maximum, and down 11.7% maximum for Part A and B respectively.

(74) This is different in magnitude to the laboratory data using grape juice, ie. equivalent to Part A in this study. The percentage changes in the laboratory ferments were over 100% for some esters. The higher alcohols and the fatty acids also increased significantly. There are a number of reasons why this may occur—juice prior history, fresh versus stored juice, the effects of fermenter geometry and so on. However it is clear that pectin plus carrageenan treatment has the potential to change the flavour volatiles of wine, which the wine maker may use to manipulate, or create or enhance flavour characteristics.

(75) TABLE-US-00010 TABLE 7 Wine heat stability result for Trial 1, Part A and Part B Demi no. 13 14 9 10 16 11 17 18 29 30 27 28 Heat test 36.7 36 44.1 43.4 43.9 44.4 42.8 39.2 52.6 36.1 32.4 31.4 (2 h) Bentonite 800 800 400 400 500 400 400 400 800 400 400 400 test (ppm)

(76) Heat stability of these ferments was tested as shown in Table 7. The control wine requires 800 mg/L of bentonite, whereas the treated wines only needed 400 mg/L in all cases apart from one (T2A); all other 8 test wines were equivalent.

(77) Trial 2

(78) Trial 1, Part A protocol was repeated with Sauvignon Blanc juice. However 2 additional conditions were included: in T2D and 2E carrageenan powder replaced carrageenan solution. The tests were set up as follows: Control—no pectin or carrageenan Treatment 2A—Addition of 1.5 g/L pectin (powder) and 0.150 g/L carrageenan and incubation for 1 minute before pectinase addition and chilling (duplicate). Treatment 2B—Addition of 1.5 g/L pectin (powder) and 0.150 g/L carrageenan and incubation for 10 minutes before pectinase addition and chilling (duplicate). Treatment 2C—Addition of 1.5 g/L pectin (powder) and 0.150 g/L carrageenan and incubation for 30 minutes before pectinase addition and chilling (duplicate). Treatment 2D—Addition of 1.5 g/L pectin (powder) and 0.250 g/L carrageenan and incubation for 10 minutes before pectinase addition and chilling (duplicate). Treatment 2E—Addition of 1.5 g/L pectin (powder) and 0.250 g/L carrageenan and Incubation for 30 minutes before pectinase addition and chilling (duplicate).

(79) Pectin and carrageenan were vigorously mixed with juice using a hand-held, electric impeller, according to the plan above. Pectinase (400 ul in 12 L) was subsequently added and the demijohns were placed at 4° C. for settling for 16 hours. Once settled, all treatments were racked, then allowed to warm to 16° C. and inoculated with EC1118 yeast at 200 mg/L. The juices were finally fermented and bottled according to the Trial 1 protocol.

(80) TABLE-US-00011 TABLE 8 Treatment data for Trial 2. The Sauvignon Blanc juices were treated by a combination of carrageenan solution and pectin powder in T2A, 2B and 2C whereas in T2D and 2E, carrageenan solution was replaced by carrageenan powder. After the treatment the juices were settled at 4° C. for 16 h. Wine yeast EC1118 was inoculated into the juices and then fermented at ambient (20° C.) temperature for 7 days. Initial Racked DJ No. Volume (L) Volume (L) Juice data Control 10 12 10.50 pH 3.24 20 12 10.60 TA 10.7 T2A 3 12 10.15 Bé 11 8 12 10.05 YAN 307 T2B 2 12 10.25 SO.sub.2 30 7 12 10.15 T2C 4 12 10.40 6 12 10.40 T2D 5 12 10.35 14 12 10.30 T2E 18 12 10.30 11 12 10.40

(81) TABLE-US-00012 TABLE 9 Fermentation data for Trial 2 after Day 7 DJ No. RS FSO.sub.2 TSO.sub.2 pH TA Alc VA 10 0.25 36 131 3.14 10.2 10.9 0.13 20 0.09 33 161 3.14 10.6 10.8 0.22 3 0.24 29 136 3.17 10.4 10.9 0.34 8 0.29 35 134 3.18 10.0 10.9 0.20 2 0.34 26 130 3.20 10.0 10.9 0.24 7 0.20 33 149 3.19 10.1 10.8 0.44 4 1.51 29 153 3.17 10.3 10.9 0.37 6 0.20 36 140 3.18 10.1 10.9 0.19 5 0.30 29 129 3.19 10.1 10.9 0.23 14 0.25 36 137 3.19 10.1 11.0 0.28 18 0.29 35 133 3.18 10.1 10.9 0.14 11 0.21 37 149 3.18 10.1 10.9 0.35

(82) TABLE-US-00013 TABLE 10 Wine analysis and the percentage changes in volatiles for Trial 2 Control 2A 2B 2C 2D 2E % 2A 2B 2C 2D 2E ETOAC 40.55 41.45 45.80 42.65 45.35 45.70 2.2 12.9 5.2 11.8 12.7 ETHEX 0.98 0.82 0.78 0.96 0.87 1.02 −15.9 −20.5 −2.1 −10.8 4.1 ETOCT 0.94 0.79 0.79 0.86 0.86 0.87 −16.0 −16.0 −8.5 −9.0 −8.0 PHETAC 0.55 0.56 0.64 0.47 0.59 0.45 2.8 16.5 −14.7 8.3 −17.4 IAMOAC 3.67 3.59 4.67 3.87 4.40 3.66 −2.2 27.1 5.3 19.9 −0.3 TOTEST 46.65 47.20 52.65 48.80 52.05 51.65 1.2 12.9 4.6 11.6 10.7 NPROH 32.70 26.25 20.65 29.65 26.90 35.30 −19.7 −36.9 −9.3 −17.7 8.0 IBJOH 20.40 20.50 22.50 17.80 20.90 19.15 0.5 10.3 −12.7 2.5 −6.1 IAMOH 142.65 137.80 131.20 130.90 130.30 133.35 −3.4 −8.0 −8.2 −8.7 −6.5 PHETOH 30.75 31.25 25.35 24.60 24.60 25.00 1.6 −17.6 −20.0 −20.0 −18.7 TOTALC 226.50 215.80 199.70 202.95 202.70 212.80 −4.7 −11.8 −10.4 −10.5 −6.0 BUT 1.29 1.40 1.21 1.43 1.34 1.56 8.1 −6.6 10.5 3.5 20.5 HEX 5.01 4.84 4.59 5.07 4.84 5.25 −3.4 −8.5 1.2 −3.4 4.7 OCT 9.81 8.90 8.61 9.38 9.44 10.22 −9.3 −12.2 −4.4 −3.8 4.1 DEC 3.24 2.58 2.58 2.95 2.94 2.96 −20.4 −20.5 −9.0 −9.4 −8.6 TOTACD 19.82 18.14 17.38 19.20 18.93 20.39 −8.5 −12.3 −3.2 −4.5 2.9

(83) Volatile data showed the sane trends as before: ie. esters increased, higher alcohols slightly decreased and total acids slightly decreased.

(84) TABLE-US-00014 TABLE 11 Heat stability data for Trial 2 DJ No. 10 20 3 8 2 7 4 6 5 14 18 11 Heat test 67.4 61.8 49.7 28.0 49.0 43.7 52.8 45.3 41.1 33.1 19.8 24.4 (2 h) Bentonite 1200 1200 700 600 800 600 600 700 500 500 600 500 test (ppm)

(85) The heat stability data shows a large drop in the bentonite requirement for hot stabilization. In the control wine the bentonite dose for stabilization reached 1200 mg/L. This could be reduced to less than half by some of the pectin plus carrageenan combination as shown in Table 11.

(86) These trials were carried out with fresh grapes or juice under winemaking conditions. A large reduction in bentonite requirement was observed using pectin plus carrageenan treatments. Changes in the volatiles distribution—although not as large as observed under small scale, laboratory conditions, was sufficient to show that these pre-treatments can be used if required to modify the flavour actives in finished wine.

Example 5—Effects of Carrageenan Dose Rate, the Contact Time Between Grape Juice and Pectin Plus Carrageenan During Wine Fermentation

(87) This Example was carried out using fresh Sauvignon Blanc grapes grown in Victorian Centre grape growing region. Free run juice was collected and combined with juice after pressing. The details of juice are shown in Table 12 below:

(88) TABLE-US-00015 TABLE 12 Sauvignon Blanc juice used in this study. Bé pH TA FS0.sub.2 TS0.sub.2 YAN Malate 12 3.25 7.77 14 41 239 4

(89) Each of 1.5 L juice was added into a 2 L bench top fermenter and mixed vigorously with pectin powder (TS 1580) plus 2% carrageenan solution (CSW-2) using the following recipe for 15 min. An untreated juice was used as the control. The control and the treated juices were mixed with 0 ppm of pectinase and then stored at 4° C. for 16 h before racking. There was no racking in Treatment 5 and 6 (T5 and T6) and therefore the added pectin and carrageenan were carried over to fermentation.

(90) TABLE-US-00016 TABLE 13 Juice treatment table. Pectin and carrageenan were mixed with juice as 20° C. using a magnetic stirrer for 15 min. Treatment Control 1 2 3 4 5 6 Pectin 0 1.5 1.5 1.5 1.5 1.5 1.5 (g/L) Carrageenan 0 0.15 0.20 0.30 0.50 0.15 0.15 (g/L) Juice 1.5 1.5 1.5 1.5 1.5 1.5 1.5 Volume (L) Volume 1.5 1.4 1.4 1.3 1.3 1.5 1.5 after racking

(91) 10% wine yeast (EC1118) slurry was inoculated into each juice once the fermenters were warmed to 16° C.

(92) Fermentation was controlled between 20-22° C. and the fermentation rate was monitored by measuring Bé (Table 14). Once Bé of each ferment reached zero, it was placed at 4° C. for 7 days. The supernatant (wine) were collected and passed through a 0.45 μm Nylon syringe filter. Heat stability tests were finally carried out using the filtered wine samples using standard test protocols.

(93) TABLE-US-00017 TABLE 14 Fermentation data for Sauvignon Blanc. Fermentation temperature was between 20-22° C. Sugar (Bé) Control 1 2 3 4 5 6 Day 0 12.0 12.0 12.0 12.0 12.0 12.0 12.0 Day 2 9.4 9.2 8.9 8.8 8.6 8.6 8.8 Day 5 2.1 2.8 2.2 2.2 2.6 2.6 2.8 Day 7 0 0 0 0.20 0 0 0

(94) The data show that the sugar utilisation was very similar during fermentation in all test cases compared to the control. All fermentations ceased after 7 days and Bé was effectively zero.

(95) TABLE-US-00018 TABLE 15 Heat stability data of the treated wine and the control wine. Duplicate samples were taken and measured in most conditions except T4 and T5. Filtration flux is ranked from fast (0) to very slow (5). Treatment Control 1 2 3 4 5 6 Filtration 5 5 3 0 0 5 5 difficulty Turbidity 33.7 9.59 2.82 2.14 1.59 1.59 1.64 (NTU) 34.7 9.32 3.04 1.49 1.48

(96) The data (Table 15) shows clearly that wine heat stability is closely related to carrageenan dosage rate. Hence increased carrageenan dosage results in more stable wine judged by the heat test (cf. T1, 2, 3 and 4 in Table 17). This is especially apparent once 0.5 g/L carrageenan was added into the juice. The resulting wine is heat stable, requires no further bentonite treatment. However, the filtration data shows that high carrageenan dose rates reduce filterability to unacceptable values. However, keeping the pectin and carrageenan in the fermentation achieves excellent heat stability as compared to juices that were racked before fermentation (T5 and T6 vs T1). Low carrageenan dose rate can even achieve heat stability and require no further post-fermentation-treatments, provided there is a long-enough contact time, such as in the example above, by leaving carrageenan in the juice during fermentation. At the same time the carrageenan treatment rates are sufficiently low to have little impact on standard filtration practice.

(97) In the claims which follow and in the preceding description of the invention, except where the context requires otherwise due to express language or necessary implication, the word “comprise” or variations such as “comprises” or “comprising” is used in an inclusive sense, i.e. to specify the presence of the stated features but not to preclude the presence or addition of further features in various embodiments of the invention.