Spent coffee grounds-derived beverage

Technical Field

The present invention relates to an alcoholic beverage derived from spent coffee grounds.

Background

Spent coffee grounds (SCG) are the residue remaining after coffee brewing and instant coffee manufacturing. Huge amounts of SCG are generated every year. SCG is discarded as food waste and this causes serious environmental hazards. The rising concerns about the negative effects of SCG on environment have resulted in the development of processing SCG for use in the food industry. However, fermentable sugars present in SCG cannot support sufficiently the growth of bacteria or yeasts.

There is therefore a need for an improved method and use of SCG.

Summary of the invention The present invention seeks to address these problems, and/or provides a novelty spent coffee grounds-derived beverage.

According to a first aspect, there is provided an alcoholic beverage comprising esters and having an ethanol content of £ 10% by volume, wherein the esters comprise at least one of: ethyl 2,4-hexadienoate and ethyl decanoate; and wherein the alcoholic beverage is an undistilled alcoholic beverage and is derived from spent coffee grounds.

The beverage may further comprise at least one ester selected from, but not limited to, ethyl (£)-4-hexenoate, isoamyl acetate, 3-methylbutyl octanoate, ethyl nonanoate, ethyl 9-decenoate, phenethyl acetate.

According to a particular aspect, the beverage may be a fermented beverage. The beverage may have a suitable ethanol content. For example, the beverage may have an ethanol content of 0.5-8% by volume.

The beverage may have any suitable pH. For example, the pH of the beverage may be 3.5-6.5. The beverage may have a suitable Brix. For example, the Brix of the beverage may be 5-26° Bx.

According to a second aspect of the present invention, there is provided a method of forming an undistilled alcoholic beverage derived from spent coffee grounds, the method comprising: hydrolysing a liquid mixture of spent coffee grounds to obtain a hydrolysed liquid mixture; adding yeast to the hydrolysed liquid mixture; and fermenting the hydrolysed liquid mixture comprising yeast at a pre- determined temperature for a pre-determined period of time to form the beverage.

In particular, the alcoholic beverage formed from the method may comprise esters, the esters comprising at least one ester selected from: ethyl 2,4-hexadienoate and ethyl decanoate.

The hydrolysing may comprise acid hydrolysing the liquid mixture. In particular, the acid hydrolysing may be with a food-grade acid. The food-grade acid may be any suitable acid, such as, but not limited to, citric acid, malic acid, lactic acid, or a combination thereof. The hydrolysing may further comprise enzyme hydrolysing following the acid hydrolysing. The enzyme hydrolysing may comprise hydrolysing the liquid mixture with any suitable enzyme. For example, the enzyme may comprise, but is not limited to, carbohydrase.

The yeast may be any suitable yeast. According to a particular aspect, the yeast may be, but not limited to, Saccharomyces yeast, non- Saccharomyces yeast, or a combination thereof. For example, the yeast may be, but not limited to, Kluyveromyces (K.) thermotolerans, Saccharomyces (S.) cerevisiae, Torulaspora (T.) delbrueckii, Pichia (P.) kluyveri, Lachancea (L.) thermotolerans, or a combination thereof. The adding may comprise adding any suitable amount of yeast. According to a particular aspect, the adding may comprise adding yeast to obtain an initial yeast count of 5-7 log CFU yeast/mL.

The pre-determined period of time may be any suitable period of time for the purposes of the present invention. According to a particular aspect, the pre-determined period of time may be 2-29 days.

The pre-determined temperature may be any suitable temperature for the purposes of the present invention. According to a particular aspect, the pre-determined temperature may be 15-37°C. According to a particular aspect, the method may further comprise adding sugar to the hydrolysed liquid mixture prior to the adding yeast to the hydrolysed liquid mixture. The sugar added may be any suitable sugar for the purposes of the present invention. In particular, the adding sugar to the hydrolysed liquid mixture may comprise adding sugar such that the hydrolysed liquid mixture has a Brix of 5-26 °Bx. According to a particular aspect, the method may further comprise heating the hydrolysed liquid mixture prior to the adding yeast to the hydrolysed liquid mixture.

The method may further comprise adding yeast extract prior to the adding yeast to the hydrolysed liquid mixture. The method may further comprise adjusting pH of the hydrolysed liquid mixture prior to the adding yeast to the hydrolysed liquid mixture. According to a particular aspect, the adding yeast may further comprise adding lactic acid bacteria. Any suitable amount of lactic acid bacteria may be added. In particular, the adding may comprise adding lactic acid bacteria to obtain an initial lactic acid bacteria count of 4-7 log CFU/mL.

The lactic acid bacteria may be any suitable lactic acid bacteria. According to a particular aspect, the lactic acid bacteria may be, but not limited to, Oenococcus (O.) oeni, Lactobacillus (L.) plantarum, or a combination thereof.

Brief Description of the Drawings

In order that the invention may be fully understood and readily put into practical effect there shall now be described by way of non-limitative example only exemplary embodiments, the description being with reference to the accompanying illustrative drawings. In the drawings:

Figure 1 shows growth of K. thermotolerans Concerto during fermentation of two types of SCG hydrolysate media - KC: K. thermotolerans Concerto with 0% yeast extract and KY: K. thermotolerans Concerto with 0.25% yeast extract. Yeast cell counts shown are the mean values of triplicate fermentations with error bars indicating the standard deviations of the mean values;

Figure 2 shows the ethanol content of the fermented SCG hydrolysate at day 22. KC: K. thermotolerans Concerto with 0% yeast extract, KY: K. thermotolerans Concerto with 0.25% yeast extract. The values shown are the mean of triplicate fermentation (n=3), with error bar representing the standard deviation of the mean a: statistical analysis using ANOVA at 95% confidence interval;

Figure 3 shows the total sugar content changes of SCG hydrolysate media fermented with K. thermotolerans Concerto. The concentrations are the mean values of triplicate fermentations (n=3) with error bars indicating the standard deviations of the mean values;

Figure 4 shows growth of S. cerevisiae MERIT during fermentation of two types of SCG hydrolysate media - SC: S. cerevisiae MERIT with 0% yeast extract and SY: S. cerevisiae MERIT with 0.25% yeast extract. Yeast cell count shown are the mean values of triplicate fermentations with error bars indicating the standard deviation of the mean values;

Figure 5 shows the ethanol content of the fermented SCG hydrolysate at day 22. SC: S. cerevisiae MERIT with 0% yeast extract and SY: S. cerevisiae MERIT with 0.25% yeast extract. The values are mean of triplicate fermentation (n=3), with error bar representing the standard deviation of the mean a: statistical analysis using ANOVA at 95% confidence interval;

Figure 6 shows the total sugar content changes of SCG hydrolysate media fermented with S. cerevisiae MERIT. SC represents S. cerevisiae MERIT with 0% yeast extract and SY represents S. cerevisiae MERIT with 0.25% yeast extract. The concentrations shown are the mean values of triplicate fermentations (n=3) with error bars indicating the standard deviation of the mean values;

Figure 7 shows growth of T. delbrueckii Biodiva during fermentation of SCG hydrolysates. TC represents T. delbrueckii Biodiva with 0% added yeast extracts and TY represents T. delbrueckii Biodiva with 0.25% added yeast extracts. Yeast cell counts are the mean values of triplicate fermentations with error bars indicating the standard deviations of the mean values;

Figure 8 shows ethanol content of the fermented SCG hydrolysate from day 0 to day 14. TC represents T. delbrueckii Biodiva with 0% yeast extracts and TY represents T. delbrueckii Biodiva with 0.25% yeast extracts. The values are the mean values of triplicate fermentations (n=3) with error bars indicating the standard deviations of the mean;

Figure 9 shows growth of P. kluyveri FrootZen during fermentation of SCG hydrolysates. PC represents P. kluyveri FrootZen with 0% yeast extracts and PY represents P. kluyveri FrootZen with 0.25% yeast extracts. Yeast cell counts are the mean values of triplicate fermentations with error bars indicating the standard deviations of the mean values;

Figure 10 shows ethanol content of the fermented SCG from day 0 to day 14. PC represents P. kluyveri FrootZen with 0% yeast extracts and PY represents P. kluyveri FrootZen with 0.25% yeast extracts. The values are the mean values of triplicate fermentations (n=3) with error bars indicating the standard deviations of the mean;

Figure 11A shows production of ethyl acetate, Figure 11 B shows production of isoamyl acetate and Figure 11C shows production of 2-phenethyl acetate, during fermentation with P. kluyveri FrootZen. The concentration is the mean values of triplicate fermentations (n = 3), with error bar representing the standard deviation of the mean. PC represents P. kluyveri FrootZen with 0% yeast extract and PY represents P. kluyveri FrootZen with 0.25% yeast extract

Figure 12A shows growth of yeast strains and Figure 12B shows growth of O. oeni strains (b) during SCG hydrolysates fermentation. C represents (·) L thermotolerans Concerto monoculture, CP represents (¾$) Co-culture of L thermotolerans Concerto and O. oeni PN4, CB represents (A) Co-culture of L. thermotolerans Concerto and O. oeni Enoferm Beta, and CL represents (T) Co-culture of L thermotolerans Concerto and O. oeni Lalvin 31. The values are the mean values of triplicate fermentations (n=3) with error bars indicating the standard deviations of the mean; and Figure 13 shows ethanol contents of fermented SCG hydrolysates. C represents (·) L. thermotolerans Concerto monoculture, CP represents ( >¾ ) Co-culture of L thermotolerans Concerto with O. oeni PN4, CB represents (A) Co-culture of L thermotolerans Concerto with O. oeni Enoferm Beta and CL represents (T) Co-culture of L. thermotolerans Concerto with O. oeni Lalvin 31. The values are the mean values of triplicate fermentations (n=3) with error bars indicating the standard deviations of the mean.

Detailed Description

As explained above, there is a need for processing spent coffee grounds (SCG) to better utilise it. Accordingly, the present invention relates to the valorization of SCG by forming a spent coffee grounds-derived beverage.

In general terms, the present invention relates to an alcoholic beverage derived from spent coffee grounds and a method of forming the same. The SCG-derived beverage may be formed from a method which is environmentally friendly and is able to valorize the SCG, thereby converting the SCG into a novelty beverage. In particular, the beverage may have a range of flavour compounds, organic acids and a certain amount of ethanol. The beverage may have a pleasant aroma due to greater amounts of fruity esters and other unique aroma compounds.

According to a first aspect, there is provided an alcoholic beverage comprising esters and having an ethanol content of £ 10% by volume, wherein the esters comprise at least one of: ethyl 2,4-hexadienoate and ethyl decanoate; and wherein the alcoholic beverage is an undistilled alcoholic beverage and is derived from spent coffee grounds.

The beverage may be any suitable spent coffee ground-derived beverage. For the purposes of the present invention, an alcoholic beverage is defined as a beverage which comprises alcohol. In particular, the alcohol may be ethanol. The beverage may have an ethanol content of ³ 0.5% by volume. For example, the beverage may have an ethanol content of 0.5-10%, 0.7-9.8%, 0.9-9.5%, 10-9.0%, 12-8.5%, 15-8.0%, 2.0- 7.0%, 2.5-6.5%, 3.0-6.0%, 3.5-5.5%, 4.0-5.0%, 4.2-4.8%, 4.3-4.5% by volume. Even more in particular, the beverage may have an ethanol content of 0.5-8.0% by volume.

According to a particular aspect, the beverage does not comprise methanol. This is advantageous since methanol may be harmful when consumed. The beverage may be an undistilled alcoholic beverage. In particular, the beverage may be a fermented beverage. A fermented beverage may be defined as a beverage which has undergone fermentation.

According to a particular aspect, the beverage may be any suitable alcoholic beverage. For example, the alcoholic beverage may include, but is not limited to, wine, ciders, beers and spirits.

The esters comprised in the beverage may confer a suitable flavour and taste to the beverage. The presence of esters in the beverage show that the beverage has undergone fermentation. In particular, the ester comprised in the beverage may impart a fruity and/or floral characteristic to the beverage. The average total ester content in the beverage may be 15-6000 pg/L. In particular, the average total ester content may be 20-6000 pg/L, 40-5800 pg/L, 60-5700 pg/L, 80- 5600 pg/L, 90-5500 pg/L, 100-5700 pg/L, 200-5400 pg/L, 220-5450 pg/L, 240-5350 pg/L, 280-5250 pg/L, 300-5250 pg/L, 400-5000 pg/L, 800-4500 pg/L, 1000-4000 pg/L, 1400-3500 pg/L, 1600-3000 pg/L, 2000-2500 pg/L. The beverage may comprise at least one of: ethyl 2,4-hexadienoate and ethyl decanoate. According to a particular aspect, the beverage may further comprise at least one ester selected from, but not limited to: ethyl (£)-4-hexenoate, isoamyl acetate, 3-methylbutyl octanoate, ethyl nonanoate, ethyl 9-decenoate, phenethyl acetate. Other esters which may be present include, but is not limited to, ethyl acetate, ethyl octanoate, ethyl hexanoate, 2-phenethyl acetate, 2-furanmethyl acetate, methyl acetate, methyl 2-furoate, methyl lactate, diacetyl, acetoin, 2-nonanone or mixtures thereof.

The beverage may have a suitable pH. For example, the pH of the beverage may be 3.5-6.5. In particular, the pH may be 3.0-5.0. The beverage may have a suitable Brix or its specific gravity equivalent. Brix is a measure of the amount of sugars in the beverage. For example, 1 °Bx refers to 1 g of sucrose in 100 g of the beverage. Accordingly, the higher the Brix, the higher the amount of sugars in the beverage which in turn may relate to a higher alcohol content in the beverage.

The Brix of the beverage may be 5-26 °Bx. For the purposes of the present invention, reference to Brix of the alcoholic beverage refers to the measure of the Brix of the spent coffee grounds comprised in the beverage. The Brix of the beverage may be 7- 25 °Bx, 10-20 °Bx, 12-18 °Bx, 13-15 °Bx. In particular, the Brix may be 5-20 °Bx. The beverage may further comprise additional additives. The additive may be any suitable additive for giving a more finished consumer product. According to a particular aspect, the additive may be, but not limited to, sweetener, flavouring, stabilizer, thickeners, colourant, preservatives, acidity regulator, or a combination thereof.

According to a second aspect of the present invention, there is provided a method of forming an undistilled alcoholic beverage derived from spent coffee grounds (SCG), the method comprising: hydrolysing a liquid mixture of spent coffee grounds to obtain a hydrolysed liquid mixture; adding yeast to the hydrolysed liquid mixture; and - fermenting the hydrolysed liquid mixture comprising yeast at a pre determined temperature for a pre-determined period of time to form the beverage.

The SCG may be any suitable spent coffee grounds and may be obtained from any suitable source. The SCG may be treated to convert the SCG into a suitable form for use in the method of the present invention. Therefore, the method may further comprise treating the SCG prior to the hydrolysing.

The treating may comprise defatting the SCG. The defatting of the SCG may be by any suitable method. According to a particular aspect, the defatting may comprise defatting the SCG with a suitable solvent such as, but not limited to, hexane, methanol, or a combination thereof. The treating may further comprise grinding the SCG into powder form. The powdered SCG may then be dissolved in a suitable solvent to form a SCG liquid mixture. For example, the solvent may be, but not limited to, water.

The concentration of the SCG in the liquid mixture may be any suitable concentration. For example, the concentration of SCG in the liquid mixture may be 2-40 w/v %. In particular, the concentration may be 2-37 w/v %, 4-35 w/v %, 6-33 w/v %, 8-31 w/v %, 10-29 w/v %, 12-27 w/v %, 14-25 w/v %, 16-23 w/v %, 18-21 w/v %, 20-37 w/v %, 18-37 w/v %, 4-30 w/v %. Even more in particular, the concentration may be 10-20 w/v%.

The treating may further comprise drying the SCG following the defatting. The drying may be under suitable conditions. In particular, the drying may comprise drying the SCG to enable the SCG to be ground into a suitable form such as a powder form.

The hydrolysing may be by any suitable method to hydrolyse and convert the insoluble macromolecules, such as but not limited to polysaccharides, into smaller soluble molecules, such as sugars, to facilitate fermentation. In particular, the hydrolysing may enable recovery of up to 30% of sugars to be recovered from the SCG. According to a particular aspect, the hydrolysing may comprise acid hydrolysing the liquid mixture. The acid hydrolysing may be by any suitable acid. In particular, the acid hydrolysing may be using a food-grade acid. For the purposes of the present invention, a food-grade acid is defined as an acid which is suitable for being consumed. The food- grade acid may be any suitable acid, such as, but not limited to, citric acid, malic acid, lactic acid, or a combination thereof.

The hydrolysing may be under suitable conditions. For example, the conditions may comprise the hydrolysing time and hydrolysing at a suitable temperature. According to a particular aspect, the acid hydrolysing may be for 10-120 min. For example, the acid hydrolysing may be for 15-110 min, 20-100 min, 30-90 min, 35-80 min, 40-70 min, 45- 60 min, 50-55 min. In particular, the acid hydrolysing may be for 40-60 min.

According to a particular aspect, the acid hydrolysing may be at a temperature of 75- 140°C. For example, the temperature may be 77-135°C, 79-130°C, 80-120°C, 90- 110°C, 100-105°C. In particular, the temperature may be 90-130°C.

The hydrolysing may further comprise enzyme hydrolysing the liquid mixture following the acid hydrolysing. The enzyme hydrolysing may comprise hydrolysing the liquid mixture with any suitable enzyme. For example, the enzyme may comprise, but is not limited to, carbohydrase. The carbohydrase may be any suitable carbohydrase, such as but not limited to, cellulase, hemicellulase, pectinase, or a combination thereof. A suitable amount of carbohydrase may be added to the acid hydrolysed liquid mixture. The enzyme hydrolysing may be under suitable conditions. For example, the enzyme hydrolysing may be for a suitable period of time. In particular, the enzyme hydrolysing may be for 2-50 hours. 4-45 hours, 6-40 hours, 8-35 hours, 10-30 hours, 12-24 hours, 15-20 hours. Even more in particular, the enzyme hydrolysing may be for 4-24 hours.

The enzyme hydrolysis may be at a suitable temperature. In particular, the enzyme hydrolysing may be at 18-60°C. For example, the temperature may be 20-57°C, 22- 55°C, 25-50°C, 30-45°C, 35-40°C. Even more in particular, the temperature may be 40-50°C.

The enzyme hydrolysis may be at a suitable pH. In particular, the enzyme hydrolysing may be at a pH of 4-8. Even more in particular, the pH may be 4-6. Following the hydrolysing, a hydrolysed liquid mixture of SCG may be formed. The hydrolysed liquid mixture may be cooled prior to the adding of yeast. Accordingly, the method may further comprise cooling the hydrolysed liquid mixture prior to the adding yeast.

According to a particular aspect, the method may further comprise adding sugar to the hydrolysed liquid mixture prior to the adding yeast to the hydrolysed liquid mixture. The addition of the sugar may be for increasing carbohydrate content in the SCG liquid mixture. The sugar added may be any suitable sugar for the purposes of the present invention. For example, the sugar added may comprise, but is not limited to, glucose, sucrose, fructose, or a combination thereof. In particular, the sugar added may be sucrose.

The adding the sugar may be to adjust the Brix of the hydrolysed liquid mixture. In particular, the Brix of the hydrolysed liquid mixture may be adjusted to 5-26 °Bx. Even more in particular, the Brix of the hydrolysed liquid mixture may be adjusted to about 15 °Bx. The method may further comprise adjusting pH of the hydrolysed liquid mixture prior to the adding yeast to the hydrolysed liquid mixture. The adjusting the pH enables the yeast to grow in the hydrolysed liquid mixture. For example, the pH may be adjusted to a pH of 3.5-6.5. In particular, the pH may be adjusted to a pH of about 5. The adjusting the pH may be by any suitable method. For example, the adjusting may comprise adding a suitable acid. The acid used for the adjusting must be an acid which is suitable for being consumed. In particular, the acid may be, but not limited to, malic acid, citric acid, lactic acid, or a combination thereof. Even more in particular, the acid may be citric acid, malic acid, or a combination thereof. According to a particular aspect, the method may further comprise heating the hydrolysed liquid mixture prior to the adding yeast to the hydrolysed liquid mixture. The heating may comprise mild pasteurization of the hydrolysed liquid mixture. The heating may extend the shelf life of the liquid mixture prior to the fermenting and may also reduce the risk of contamination during the fermenting. In particular, the heating may eliminate or reduce indigenous microbial activity in the liquid mixture.

The heating may be carried out under suitable conditions. In particular, the heating may be under suitable conditions. For example, the heating may be carried out at a temperature of about 40-80°C. In particular, the temperature may be about 60-80°C. Even more in particular, the temperature may be about 60°C. The heating may be carried out for a suitable period of time. For example, the heating may be for 10-75 minutes. In particular, the heating may be for about 10-60 minutes, 15-45 minutes, 20-40 minutes, 25-30 minutes. Even more in particular, the heating may be for about 30 minutes.

The heating may be by suitable means. For example, the heating may be in a water bath.

The method may further comprise cooling the hydrolysed liquid mixture following the heating. The cooling may comprise cooling the hydrolysed liquid mixture to room temperature.

The adding may comprise adding any suitable yeast. According to a particular aspect, the yeast may be, but not limited to, Saccharomyces yeast, non- Saccharomyces yeast, or a combination thereof. For example, the yeast may be, but not limited to, Kluyveromyces (K.) thermotolerans, Saccharomyces (S.) cerevisiae, Torulaspora (T.) delbrueckii, Pichia (P.) kluyveri, Lachancea (L.) thermotolerans, or a combination thereof. In particular, the yeast may be K. thermotolerans Concerto, S. cerevisiae MERIT, T. delbrueckii Biodiva, P. kluyveri FrootZen, O. oeni Lalvin 31, O. oeni PN4, O. oeni Enoferm Beta, or a combination thereof.

The adding may comprise adding any suitable amount of yeast. For example, the amount of yeast added may be 1-9 log CFU/mL. In particular, the amount may be about 4-7 log CFU/mL. Even more in particular, the amount may be about 5-7 log CFU/mL. According to a particular aspect, the adding may comprise adding yeast to obtain an initial yeast count of 5 log CFU yeast/mL.

According to a particular aspect, the method may further comprise adding nitrogen compounds to the hydrolysed liquid mixture prior to the adding yeast to the hydrolysed liquid mixture. The addition of nitrogen compounds may be for increasing nitrogen content in the hydrolysed liquid mixture. The nitrogen compound added may be any suitable substance containing available nitrogen. For example, the nitrogen compound added may comprise, but it is not limited to, ammonia, free amino acids, or a combination thereof. In particular, the nitrogen compound may be yeast extracts.

The subsequently added yeasts may convert the yeast extract components into volatile compounds, such as esters. Accordingly, the addition of yeast extracts may enhance the pleasant aroma of the final beverage formed from the method.

According to a particular aspect, the adding yeast may further comprise adding lactic acid bacteria. Any suitable amount of lactic acid bacteria may be added. In particular, the adding may comprise adding lactic acid bacteria to obtain an initial lactic acid bacteria count of 4-7 log CFU/mL. Even more in particular, the adding may comprise adding lactic acid bacteria to obtain an initial lactic acid bacteria count of 4-7 log CFU/mL, 5-7 log CFU/mL, or 6-7 log CFU/mL.

The lactic acid bacteria may be any suitable lactic acid bacteria. According to a particular aspect, the lactic acid bacteria may be, but not limited to, Oenococcus (O.) oeni, Lactobacillus (L.) plantarum, or a combination thereof. The fermenting may be carried out under suitable conditions. For example, the fermenting may be at a pre-determined temperature for a pre-determined period of time. The pre-determined period of time may be any suitable period of time for the purposes of the present invention. According to a particular aspect, the pre-determined period of time may be 2-29 days. In particular, the pre-determined period of time may be about 3-25 days, 5-22 days, 7-20 days, 9-18 days, 10-20 days, 12-18 days, 14-15 days. Even more in particular, the pre-determined period of time may be about 14-22 days. According to a particular aspect, the pre-determined period of time may be about 14 days. According to another particular aspect, the pre-determined period of time may be about 22 days.

The pre-determined temperature may be any suitable temperature for the purposes of the present invention. According to a particular aspect, the pre-determined temperature may be 15-37°C. In particular, the pre-determined temperature may be 15-37°C, 17- 35°C, 20-30°C, 22-25°C. Even more in particular, the pre-determined temperature may be about 20-30°C. According to a particular aspect, the pre-determined temperature may be about 30°C. According to another particular aspect, the pre-determined temperature may be about 20°C.

According to a particular aspect, the fermenting may be conducted statically, such that the liquid mixture is not subjected to any shaking or movement. During the fermenting, the yeasts metabolise the fermentable sugars and convert the existing substances such as aldehydes and acids into esters, or fructose and glucose into ethanol, thereby producing an alcoholic beverage and enriching the aroma profile of the beverage.

The method of the present invention provides a method of preparing a spent coffee grounds-derived beverage which may have lower alcohol content, a pleasant taste and aroma. The beverage formed from the method of the present invention utilises yeast and optionally lactic acid bacteria. Use of yeast in the fermenting may generate a large amount of aroma compounds that make the spent coffee ground transform into a beverage with improved taste and highly fruity aroma. In particular, the alcoholic beverage formed from the method may comprise esters, wherein the esters comprise at least one ester selected from: ethyl 2,4-hexadienoate and ethyl decanoate. The beverage formed from the method may further comprise at least one ester selected from, but not limited to: ethyl (£)-4-hexenoate, isoamyl acetate, 3-methylbutyl octanoate, ethyl nonanoate, ethyl 9-decenoate, phenethyl acetate. Other esters which may be present include, but is not limited to, ethyl acetate, ethyl octanoate, ethyl hexanoate, 2-phenethyl acetate, 2-furanmethyl acetate, methyl acetate, methyl 2- furoate, methyl lactate, diacetyl, acetoin, 2-nonanone, or mixtures thereof.

The flavour/aroma may be considered to be the most important attribute to define the quality of an alcoholic beverage such as wine. The wine aroma comes from the volatile aroma compounds that may be generated during yeast fermentation. During fermentation, yeasts generate a large number of aroma compounds such as volatile fatty acids, higher alcohols, esters that greatly influence the aroma of wine. Hence, the choice of yeast and fermentation conditions that is suitable for the type of medium and wine style are important.

Having now generally described the invention, the same will be more readily understood through reference to the following examples which are provided by way of illustration, and are not intended to be limiting.

EXAMPLE

Preparation of SCG hydrolysates

To obtain SCG hydrolysates, SCG were defatted with hexane (1:10, w/v) and dried in a fume hood overnight. The defatted SCG were ground into fine powder and dissolved in water (15%, w/v). These liquid mixtures were sequentially hydrolyzed by acid (citric acid, 200 mM, 121 °C, 1 h) and enzyme (Viscozyme®L, 6%, v/w, 50°C, 24 h, pH 5). The SCG hydrolysates were cooled to room temperature before adding 93.4 g/L sucrose into the SCG with the final °Brix value of 15. Yeast extract (either 0% or 0.25%, w/v) was added into the SCG medium before pH values were adjusted to 5.

The hydrolysates were subsequently pasteurized at 60°C for 30 min. The effectiveness of pasteurization was checked by plate counting, as is known in the art. Then pure yeast was added to the pasteurized SCG medium with an initial cell count of about 5 log CFU/mL. Fermentation was conducted statically at 20°C for 22 days. The final product obtained was then further analyzed for the sugars, ethanol, organic acids, phenolics, amino acids and aroma contents.

Example 1 - Formation of beverage from a non-Saccharomyces yeast

Two types of SCG hydrolysate media (one with added 0% yeast extract and one with 0.25% yeast extract) were prepared as described above. As mentioned above, the pH of the media was adjusted to pH 5 and pasteurized at 60°C for 30 min, followed by cooling to room temperature.

Fermentation of SCG was performed by inoculating K. thermotolerans Concerto to obtain an initial cell population of approximately 5 log CFU/mL. Mixture of yeast and SCG medium were cultured statically at 20°C for 22 days. Both fermented SCG media were analysed for sugars, ethanol, organic acids, phenolics, amino acids and aroma compound contents.

Results

Yeast growth As can be seen in Figure 1, K. thermotolerans Concerto grew well in the SCG media with an overall cell population increase of 1.95-1.98 log CFU/mL, especially with the addition of yeast extract.

Ethanol content

Ethanol was the main compound produced by the fermentation of SCG medium with K. thermotolerans Concerto. As shown in Figure 2, the ethanol content of final SCG beverages after 22 days of fermentation with 0% yeast extract and 0.25% yeast extract was 4.41% and 4.58%, respectively.

Non-volatile components

Besides ethanol, other compounds such as the amount of organic acids, phenolics, sugars, and amino acids were also measured. Table 1 provides the values of the different components measured. Day 0_ Day 22

Compound (g/L)

Pre-treated SCG Concerto (KC) Concerto + YE (KY)

Organic acids

Citric acid 34.87 ± 0.78a 36.98 ± 0.087b 35.42 ± 0.79ab Malic acid 0.80 ± 0.01c 0.27 ± 0.02b 0.22 ± 0.00a Quinic acid 0.71 ± 0.01 a 1.75 ± 0.14c 1.56 ± 0.06b Succinic acid 1.84 ± 0.01a 1.74 ± 0.12a 2.23 ± 0.17b Acetic acid 0.24 ± 0.02a 0.32 ± 0.01 b 0.55 ± 0.02c

Alkaloids (mg/L)

Trigonelline 451.87 ± 13.14b 451.87 ± 13.14b 376.11 ± 34.97a

Theobromine 93.30 ± 2.25b 7.36 ± 1.00a 18.89 ± 0.16a

Theophylline 28.82 ± 3.13c 9.12 ± 0.25a 8.64 ± 0.92a

Caffeine 1.25 ± 0.01 b 1.18 ± 0.02ab 1.14 ± 0.05a

Phenolic acids (mg/L)

Chlorogenic acids 133.73 ± 2.54b 43.31 ± 1.27a 43.58 ± 1.09a Caffeic acid 606.06 ± 9.68a 655.87 ± 3.78b 643.25 ± 7.20b Ferulic acid 9.99 ± 1.55a 20.34 ± 1.09b 19.53 ± 1.85b p-Coumaric acid 4.26 ± 0.63a 9.19 ± 0.81 c 9.46 ± 0.18c

Sugars

Fructose 25.18 ± 0.04b 4.17 ± 0.02a 4.26 ± 0.03a

Glucose 71.35 ± 0.06b 4.90 ± 0.02a 4.90 ± 0.03a

Sucrose 12.38 ± 0.01 b 2.38 ± 0.05a 2.34 ± 0.01a

Mannose 21 .67 ± 0.00b 4.25 ± 0.00a 4.10 ± 0.01a

Galactose 23.02 ± 0.00b 4.54 ± 0.01 a 4.53 ± 0.01a

Arabinose 2.44 ± 0.05b 2.33 ± 0.03a 2.41 ± 0.05ab

Total sugar 155.59 ± 1.66b 22.56 ± 0.14a 22.56 ± 0.14a

SCG: spent coffee grounds; YE: adding 0.25% yeast extract; KC: K. thermotolerans Concerto with 0% yeast extract, KY: K. thermotolerans Concerto with 0.25% (w/v) yeast extract; a, b, c: statistical analysis using ANOVA (n=3) at 95% confidence interval. Same letters indicate no significant difference between samples

Table 1: Measured components contained in fermented SCG alcoholic beverage formed from fermentation of SCG hydrolysate using K. thermotolerans Concerto Several organic acids such as citric acid, malic acid, quinic acid, succinic acid, and acetic acid were quantified (see Table 1). The amounts of citric acid (36.98 g/L), quinic acid (1.75 g/L), and acetic acid (0.32 g/L) increased significantly after fermentation, while the amount of malic acid decreased significantly after fermentation. Moreover, the production of succinic acid (2.23 g/L) and acetic acid (0.55 g/L) were further increased when the SCG media was supplemented with yeast extract.

Phenolic compounds and alkaloids were also quantified, as provided in Table 1. For alkaloids, theophylline was produced significantly after fermentation while others remained stable. For phenolic acids, caffeic acid, ferulic acid and p-coumaric acid were generated significantly after fermentation. However, the supplementation of yeast extract to the SCG hydrolysate showed no effect on the generation of phenolic acids.

The SCG hydrolysates contained various sugars including fructose, glucose, sucrose (added), mannose, galactose and arabinose. The total sugar content declined significantly from 150 g/L to around 22 g/L, as can be seen in Figure 3. Almost all the sugars were significantly consumed by yeast during fermentation, and this consumption was accelerated in when the SCG hydrolysate media was supplemented with yeast extract.

The concentrations of amino acids before and after SCG fermentation with K. thermotolerans Concerto was also measured. It was found that after fermentation, most amino acids, except for proline and histidine, significantly declined to low levels. The consumed amino acids played important roles in production of aroma compounds in the formed alcoholic beverage.

Volatile compounds The changes in the volatile composition of the SCG hydrolysate pre and post fermentation are shown in Table 2. Among the volatile compounds measured and quantified, esters were the main contributors to the fruity aroma of the alcoholic beverage formed.

Phenylethyl alcohol, ethyl acetate, ethyl (£)-4-hexenoate, ethyl octanoate, ethyl decanoate, isoamyl acetate played significant roles in providing a fruity aroma in the fermented SCG hydrolysate medium. Production of several esters was boosted by the addition of yeast extract.

LRI: linear retention index; determined on a DB-FFAF column relative to C7-C40 hydrocarbons; YE: adding 0.25% yeast extract; KC: K. thermotolerans Concerto without yeast extract, KY: K. thermotolerans Concerto with 0.25% (w/v) yeast extract; Values are the mean of triplicate fermentations (± standard deviation); a,b,c Statistical analysis using ANOVA (n=3) at 95% confidence interval. Same letter indicates no significant difference between samples

Table 2: Selected volatile compounds and their concentrations ^g/L) before and after fermentation with K. thermotolerans Concerto

In conclusion, SCG hydrolysates fermented with non-Saccharomyces yeast K. thermotolerans Concerto may be used for producing SCG-derived alcoholic beverage with health benefit compounds and a pleasant aroma profile.

Example 2 - Formation of beverage from a Saccharomyces yeast Two types of SCG hydrolysate media (one with added 0% yeast extract and one with 0.25% yeast extract) were prepared as described above. As mentioned above, the pH of the media was adjusted to pH 5 and pasteurized at 60°C for 30 min, followed by cooling to room temperature. Fermentation of SCG was performed by inoculating SCG hydrolysates with S. cerevisiae MERIT to obtain an initial cell population of approximately 5 log CFU/mL. Mixture of yeast and SCG medium were cultured statically at 20°C for 22 days. Both fermented SCG media were analysed for sugars, ethanol, organic acids, phenolics, amino acids and aroma compound contents. Results

Yeast growth

As can be seen in Figure 4, S. cerevisiae MERIT grew well in the SCG media with an overall cell population increase of 2.40-2.81 log CFU/mL from day 0 to day 22, and this was further improved by the addition of yeast extract. Ethanol content

Ethanol was the main compound produced by the fermentation of SCG medium with S. cerevisiae MERIT. As shown in Figure 5, the ethanol content of final SCG beverages after 22 days of fermentation with 0% yeast extract and 0.25% yeast extract was 4.75% and 4.64%, respectively. Non-volatile components

Besides ethanol, other compounds such as the organic acids, phenolics, sugars, and amino acids produced were also measured. Table 3 provides the values of the different components measured.

Several organic acids such as citric acid, malic acid, quinic acid, succinic acid, and acetic acid were quantified (see Table 3) on day 0 and day 22. The amount of quinic acid (1.75 g/L) and acetic acid (0.61 g/L) increased significantly after fermentation, while the amount of malic acid declined significantly after fermentation. Moreover, the generation of succinic acid (2.56 g/L) and acetic acid (0.77 g/L) were further increased with the supplementation of yeast extract in the SCG hydrolysate, while the concentration of malic acid further declined with the addition of yeast extract.

Compound Day 0 _ Day 22 _

Merit + YE

(g/L) Pre-treated SCG Merit (SC) (SY)

Organic acids

Citric acid 34.87 ± 0.78a 36.05 ± 1.28a 36.01 ± 0.34a Malic acid 0.80 ± 0.01 c 0.37 ± 0.02b 0.21 ± 0.00a Quinic acid 0.71 ± 0.01 a 1.75 ± 0.05b 1.84 ± 0.05b Succinic acid 1.84 ± 0.01 a 1.92 ± 0.10a 2.56 ± 0.06b Acetic acid 0.24 ± 0.02a 0.61 ± 0.02b 0.77 ± 0.01 c Alkaloids Trigonelline 0.27 ± 0.00a 0.28 ± 0.00a 0.25 ± 0.01 a Theobromine 0.02 ± 0.01 b 0.01 ± 0.00a 0.01 ± 0.00 Theophylline 0.02 ± 0.01 a 0.08 ± 0.00c 0.05 ± 0.02b Caffeine 3.45 ± 0.02a 3.58 ± 0.05a 3.46 ± 0.03a

Phenolic acids

Chlorogenic acids 0.03 ± 0.01 a 0.02 ± 0.00a 0.02 ± 0.00a Caffeic acid 0.26 ± 0.02a 0.60 ± 0.00b 0.51 ± 0.02b Ferulic acid 0.01 ± 0.00a 0.02 ± 0.00b 0.02 ± 0.01 b p-coumaric acid 0.00 ± 0.00a 0.01 ± 0.00b 0.01 ± 0.00b

Sugars

Fructose 25.18 ± 0.04b 4.38 ± 0.00a 4.37 ± 0.05a

Glucose 71.35 ± 0.06c 4.29 ± 0.03b 3.09 ± 0.06a

Sucrose 12.38 ± 0.01 b 2.21 ± 0.04a 2.46 ± 0.00a

Mannose 21.67 ± 0.00b 4.25 ± 0.00a 4.21 ± 0.00a

Galactose 23.02 ± 0.00c 4.34 ± 0.02b 3.76 ± 0.00a

Arabinose 2.44 ± 0.05b 2.50 ± 0.04b 2.22 ± 0.00a

Total sugar 155.59 ± 1.66b 21 .96 ± 0.23a 20.11 ± 0.11 a

SCG: spent coffee grounds; YE: adding 0.25% yeast extract; SC represents S. cerevisiae MERIT without yeast extract and SY represents S. cerevisiae MERIT with 0.25% (w/v) yeast extract a, b, c: statistical analysis using ANOVA (n=3) at 95% confidence interval. Same letters indicate no significant difference between samples

Table 3: Measured components contained in fermented SCG alcoholic beverage formed from fermentation of SCG hydrolysate using S. cerevisiae MERIT

Phenolic compounds and alkaloids were also quantified, as provided in Table 3. For alkaloids, theophylline was produced significantly after fermentation while the amount of theobromine decreased significantly after fermentation with others remaining stable. The addition of yeast extract to the SCG hydrolysate only affected the decline of theophylline. For phenolic acids, caffeic acid, ferulic acid and p-coumaric acid were generated significantly after fermentation. However, the addition of yeast extract showed no effect on the generation of phenolic acids.

Various sugar profiles in the SCG hydrolysates were measured. In particular, the amounts of fructose, glucose, sucrose (added), mannose, galactose and arabinose were quantified. The total sugar content declined significantly from 155.59 g/L to around 21 g/L, as can be seen in Figure 6. All the sugars were significantly consumed by yeast during fermentation, regardless of whether the SCG hydrolysate was supplemented with or without yeast extract. However, the consumption of glucose, galactose and arabinose decreased significantly when yeast extract was added. The concentration of amino acids before and after SCG fermentation with S. cerevisiae MERIT was also measured. It was found that the addition of yeast extract enriched amino acids contents of SCG hydrolysate. In total, eighteen kinds of amino acids were quantified. After fermentation, most of the amino acids, except for histidine, significantly decreased to low levels. The consumed amino acids played important roles in the production of aroma compounds in the formed alcoholic beverage. In particular, the content of amino acids in the final SCG beverages remained in the range of 0.08 - 73.01 mg/L.

Volatile compounds

The changes in the volatile composition of SCG hydrolysate before and after fermentation are shown in Table 4. Among the volatile compounds measured and quantified, esters were the main contributors to the fruity aroma of the alcoholic beverage formed. Production of 3-methylbutyl octanoate, ethyl acetate, ethyl hexanoate, ethyl (E)-4-hexenoate, ethyl octanoate, ethyl 2,4-hexadienoate, ethyl nonanoate, ethyl decanoate, ethyl 9-decenoate, isoamyl acetate and phenethyl acetate played significant roles in contributing to the fruity aroma of the fermented SCG hydrolysate medium.

The addition of yeast extract had a positive effect on the production of some volatile compounds including the production of phenylethyl alcohol, ethyl octanoate, ethyl 9- decencate, isoamyl acetate and phenethyl acetate, as seen from Table 4. The high amounts of these volatile compounds enriched the aroma of the formed SCG-derived beverage.

LRI: linear retention index; determined on a DB-FFAF column relative to C7-C40 hydrocarbons; YE: adding 0.25% yeast extract; SC represents S. cerevisiae MERIT without yeast extract and SY represents S. cerevisiae MERIT with 0.25% yeast extract. Values are the mean of triplicate fermentations (± standard deviation); a,b,c,d Statistical analysis using ANOVA (n=3) at 95% confidence interval. Same letter indicates no significant difference between samples

Table 4: Selected volatile compounds and their concentrations ^g/L) before and after fermentation with S. cerevisiae MERIT

In conclusion, SCG hydrolysates fermented with Saccharomyces yeast S. cerevisiae MERIT may be used for producing SCG-derived alcoholic beverage with a pleasant aroma profile. Example 3 - Formation of beverage from another non -Saccharomyces yeast

Two types of SCG hydrolysates were prepared as described above, with one having 0% yeast extract and the other having 0.25% (w/v) yeast extract. As mentioned above, pH was adjusted to 5 using 10 M NaOH and subsequently pasteurized at 60°C for 30 min, followed by cooling to room temperature.

Fermentation of the hydrolysates was performed by inoculating the hydrolysates with Torulaspora delbrueckii Biodiva (Lallemand Inc, Brooklyn Park, Australia) with an initial cell population of approximately 5 log CFU/mL. The fermentation was terminated after static incubation for 14 days at 20°C. Sugars, ethanol, organic acids, phenolics, amino acids, and volatiles in the fermented media were analysed.

Results

Yeast growth

As can be seen in Figure 7, T. delbrueckii Biodiva grew well in the SCG hydrolysates with increased cell populations of 1.74-2.43 log CFU/mL and the yeast population was markedly increased when yeast extracts were added.

Ethanol content

The production of ethanol increased along with the progression of fermentation and reached a plateau by day 10 in the presence of added yeast extracts but ethanol production continued until day 14 of termination for the hydrolysates without added yeast extracts. As seen in Figure 8, the final ethanol content of fermented SCG hydrolysates were 3.65% (v/v, with 0% yeast extracts) and 3.75% (v/v, with 2.5% yeast extracts). Yeast extracts addition markedly enhanced ethanol production during fermentation from day 2 to day 10.

Non-volatile components Besides ethanol, other compounds such as the organic acids, phenolics, sugars, and alkaloids produced were also measured. Table 5 provides the values of the different components measured. Day 0 Day 14

Compound Biodiva + YE

Biodiva (TC)

SCG hydrolysate (TY)

Sugars (g/L)

Fructose 14.41 ± 0.27b 0.00 ± 0.00a 0.00 ± 0.00a

Glucose 26.37 ± 0.47b 0.00 ± 0.00a 0.00 ± 0.00a

Sucrose 8.06 ± 0.06b 0.33 ± 0.01a 0.30 ± 0.00a

Mannose 7.69 ± 0.19b 2.32 ± 0.06a 2.46 ± 0.07a

Galactose 14.94 ± 0.20b 7.63 ± 0.51a 7.26 ± 0.30a

Arabinose 2.49 ± 0.30a 2.27 ± 0.03a 2.29 ± 0.05a

Total 74.98 ± 1.23b 12.74 ± 1.11a 12.51 ± 0.74a

Organic acids (g/L)

Citric acid 38.35 ± 0.13b 36.29 ± 0.02a 36.96 ± 0.04ab a-Ketoglutaric acid (mg/L) 30.89 ± 2.40c 23.05 ± 0.53b 16.59 ± 0.61a Malic acid 0.97 ± 0.05b 0.29 ± 0.01a 0.24 ± 0.01a Succinic acid 1.83 ± 0.12a 2.40 ± 0.06b 3.05 ± 0.00c Lactic acid 0.09 ± 0.00a 0.55 ± 0.04b 0.71 ± 0.05c Acetic acid 0.04 ± 0.00a 0.58 ± 0.02b 0.57 ± 0.02b

Phenolic acids (mg/L)

Chlorogenic acids 120.86 ± 2.27b 92.51 ± 5.09a 100.69 ± 4.66ab Caffeic acid 469.88 ± 8.27a 874.83 ± 13.01b 896.66 ± 30.67b Ferulic acid 18.64 ± 0.85a 18.71 ± 0.70a 19.41 ± 0.67a p-Coumaric acid 7.40 ± 0.21a 7.13 ± 0.29a 7.74 ± 0.13a Alkaloids (mg/L) Trigonelline 523.19 ± 15.71a 699.28 ± 6.07b 725.95 ± 31 53b Theobromine 126.01 ± 3.33b 103.18 ± 2.90a 108.20 ± 4.63ab Theophylline 73.23 ± 2.17b 54.87 ± 1.89a 57.93 ± 2.48a Caffeine 1386.29 ± 39.10a 1328.01 ± 47.39a 1435.86 ± 25.26a

Note: SCG: spent coffee grounds; TC: T. delbrueckii Biodiva without yeast extracts; TY: T. delbrueckii Biodiva with 0.25% (w/v) yeast extracts a, b, c: Statistical analysis using ANOVA (n=3) at 95% confidence interval. Same letters indicate no significant difference between samples.

Table 5: Selected non-volatile compounds and their concentrations before and after fermentation with T. delbrueckii Biodiva

Sugars such as fructose, glucose, sucrose (added), mannose, galactose and arabinose were detected in the SCG hydrolysates. The total sugar contents declined significantly from 74.98 g/L to around 12.74 g/L after fermentation. All sugars were significantly consumed by yeast during fermentation, accelerated by the addition of yeast extracts. Among the detected organic acids (citric acid, a-ketoglutaric acid, malic acid, succinic acid, lactic acid and acetic acid), succinic acid (2.40 g/L), lactic acid (0.55 g/L) and acetic acid (0.71 g/L) increased significantly, while malic acid decreased significantly after fermentation. Moreover, the generation of succinic acid (3.05 g/L) was further increased with addition of yeast extracts.

Phenolic acids and alkaloids were also quantified, as shown in Table 5. For phenolic acids, caffeic acid was increased significantly while chlorogenic acid decreased after fermentation. Ferulic acid and p-coumaric acid kept stable before and after fermentation. Supplementation of yeast extracts had a positive effect on the generation of caffeic acid while no significant effects were observed on other phenolic acids. Among the four alkaloids, trigonelline was produced significantly after fermentation while others decreased slightly. Amino acids were taken up by the T. delbrueckii Biodiva when compared to the contents of amino acids before and after fermentation. Eighteen kinds of amino acids were quantified. The addition of yeast extracts increased the amount of amino acids in SCG hydrolysates. In general, a decreasing trend in the contents of most amino acids was observed. After fermentation, most amino acids except aspartic acid, glutamic acid and alanine significantly decreased to low levels. Major utilization of the amino acids corresponded to the high levels of volatile compounds generated in the alcoholic beverage (Table 6). The contents of amino acids in the final SCG beverages ranged from 1.22-126.14 mg/L.

Volatile components The identified volatile compounds of SCG hydrolysates before and after fermentation are shown in Table 6. Volatile acids (6), alcohols (3), esters (13), aldehydes (3), furan (3) and other types of volatile compounds (3) were detected. Among these volatile compounds, esters were the largest group and played a significant role in the pleasant and fruity aroma in the formed beverage, including ethyl acetate, ethyl hexanoate, ethyl (£)-4-hexenoate, ethyl octanoate, ethyl decanoate, ethyl (Z)-4-decenoate, ethyl dodecanoate, ethyl 2,4-hexadienoate, Isoamyl acetate, propyl acetate, octyl acetate, 2- phenethyl acetate, and 2-phenethyl propionate. Moreover, the production of several esters was increased with addition of yeast extracts. In particular, ethyl dodecanoate was only generated in the SCG hydrolysate with added yeast extracts. Also, three new esters (ethyl dodecanoate, ethyl (Z)-4-decenoate, octyl acetate, phenethyl propionate) were detected, which were not detected in the beverages of Examples 2 and 3. Day 0 Day final

Identific

Compound ation LRI Pre-Treated SCG SCG beverage methods

Biodiva + YE

0% YE_ 0.25% YE_ Biodiva (TC) (TY)

Acids

250.85 ± 242.01 ± 158.238 ± 195.70 ±

Acetic acid MS, LRI 1463 25.71b 19.59b 12.12a 13.67a

102.74 ± 100.99 ±

3-Methylbutanoic acid MS, LRI 1611 17.81b 15.71 b 0.00 ± 0.00a 0.00 ± 0.00a Pentanoic acid MS, LRI 1623 0.00 ± 0.00a 0.00 ± 0.00a 48.69 ± 3.91 b 46.51 ± 7.99b

4-Hexenoic acid MS, LRI 1918 0.00 ± 0.00a 0.00 ± 0.00a 26.12 ± 2.74b 27.75 ± 0.78b 145.32 ± 148.73 ±

Hexanoic acid MS, LRI 1834 7.45a 17.63a 0.00 ± 0.00b 0.00 ± 0.00b

128.89 ±

Benzoic acid MS, LRI 2452 25.52c 36.98 ± 3.34b 0.00 ± 0.00a 0.00 ± 0.00a

Alcohols

8058.07 ± 8077.29 ±

Ethanol MS, LRI N.A. 0.00 ± 0.00a 0.00 ± 0.00a 1010.54b 622.19b

909.25 ±

2-Phenylethyl alcohol MS, LRI 1934 34.27 ± 2.70a 40.94 ± 5.17a 861.22 ± 20.02b 61.06b

183.43 ±

1-Pentanol MS, LRI 1228 0.00 ± 0.00a 0.00 ± 0.00a 172.18 ± 14.38b 10.44b Esters

277.12 ±

Ethyl acetate MS, LRI N.A. 0.00 ± 0.00a 0.00 ± 0.00a 215.30 ± 9.80b 13.11 c Ethyl hexanoate MS, LRI 1252 0.00 ± 0.00a 0.00 ± 0.00a 59.30 ± 8.92b 88.97 ± 3.88c Ethyl (E)-4- hexenoate MS, LRI 1298 0.00 ± 0.00a 0.00 ± 0.00a 56.80 ± 8.15b 82.16 ± 10.35c 141.92 ±

Ethyl octanoate MS, LRI 1447 0.00 ± 0.00a 0.00 ± 0.00a 66.95 ± 6.80b 18.68b 292.67 ±

Ethyl decanoate MS, LRI 1653 0.00 ± 0.00a 0.00 ± 0.00a 84.82 ± 9.57b 31.48c Ethyl (Z)-4-decenoate MS, LRI 1601 0.00 ± 0.00a 0.00 ± 0.00a 10.05 ± 0.96b 13.80 ± 0.30c Ethyl dodecanoate MS, LRI 1829 0.00 ± 0.00a 0.00 ± 0.00a 0.00 ± 0.00a 12.43 ± 1.18b Ethyl 2,4- 106.50 ± hexadienoate MS, LRI 1509 0.00 ± 0.00a 0.00 ± 0.00a 85.51 ± 5.52b 10.08c 144.90 ±

Octyl acetate MS, LRI 1477 0.00 ± 0.00a 0.00 ± 0.00a 66.95 ± 6.80b 17.49c

2-Phenethyl acetate MS, LRI 1817 0.00 ± 0.00a 0.00 ± 0.00a 33.37 ± 2.57b 42.61 ± 6.57c

2-Phenethyl propionate MS, LRI 1882 0.00 ± 0.00a 0.00 ± 0.00a 24.53 ± 0.96b 38.35 ± 5.33c Aldehydes

127.92 ±

Benzaldehyde MS, LRI 1540 21.65b 125.21 ± 4.36b 73.53 ± 4.86a 80.83 ± 13.49a

Benzeneacetaldehyde MS, LRI 1629 0.00 ± 0.00a 0.00 ± 0.00a 30.41 ± 3.59b 50.95 ± 0.04c 1425.06 ± 1362.87 ±

3-Furaldehyde MS, LRI 1488 209.81 a 264.83a 23.168 ± 2.09b 21.48 ± 4.27b

Furan

195.44 ± 216.44 ±

2-Pentylfuran MS, LRI 1247 15.12b 15.55b 0.00 ± 0.00a 0.00 ± 0.00a

2-Formyl-5- 602.72 ± 575.27 ± methylfuran MS, LRI 1574 59.27b 76.01 b 11.29 ± 1.15a 8.12 ± 0.51 a

2-Vinylfuran MS, LRI 2001 15.15 ± 1.80b 11.72 ± 2.33a 11 .15 ± 0.63a 10.07 ± 0.51 a Phenolics Guaiacol MS, LRI 1892 12.70 ± 2.23c 10.23 ± 1.20ab 7.06 ± 1.09a 7.39 ± 1.00a Others

99.54 ±

2,4-Decadienal MS, LRI 1866 10.02b 92.14 ± 10.29b 0.00 ± 0.00a 0.00 ± 0.00a

3-Acetylpyrrole MS, LRI 1992 20.56 ± 2.79b 19.48 ± 1.55b 12.94 ± 1.87a 10.92 ± 1.67a

LRI: linear retention index; determined on a DB-FFAF column relative to C7-C40 hydrocarbons; YE: adding 0.25% yeast extract; TC: T. delbrueckii Biodiva without yeast extracts; TY: T. delbrueckii Biodiva with 0.25% (w/v) yeast extracts. Values are the mean of triplicate fermentations (± standard deviation); a,b,c Statistical analysis using ANOVA (n=3) at 95% confidence interval. Same letter indicates no significant difference between samples.

Table 6: Selected volatile compounds and their concentrations ^g/L) before and after fermentation with T. delbrueckii Biodiva

Example 4 - Formation of beverage from another non -Saccharomyces yeast

The preparation of SCG hydrolysates was as described in Example 3. Pasteurized SCG hydrolysates were inoculated with P. kluyveri FrootZen (Chr. Hansen, Horsholm, Denmark) with an initial cell population of approximately 5 log CFU/mL and was statically fermented at 20°C for 14 days. Analyses were conducted on the sugars, ethanol, organic acids, phenolics, amino acids, and volatiles contents as described in Example 3.

Yeast cell growth As seen in Figure 9, P. kluyveri FrootZen grew well in the SCG hydrolysates with increased cell populations by 2.28-2.46 log CFU/mL and it markedly increased when yeast extracts were added.

Ethanol content

The content of ethanol (1.98%) in the SCG hydrolysates with addition of 0.25% yeast extract was higher than that (1.47%) in the SCG hydrolysates without yeast extracts. Different alcohol contents were detected from day 2 to day 14 ranging from 0.2% to 1.98%, as seen in Figure 10.

Non-volatile components

Non-volatile compounds containing sugars, organic acids, phenolic acids, and alkaloids, were measured and their amounts are as shown in Table 7.

Sugars including fructose, glucose, sucrose (added), mannose, galactose and arabinose were detected in the SCG hydrolysates. The total sugar contents declined significantly from 74.98 g/L to around 20.49-35.06 g/L after fermentation. All sugars were significantly consumed by yeast during the fermentation, accelerated by the addition of yeast extracts. Day 0 _ Day 14 _

Compound FrootZen + YE

Pre-treated SCG FrootZen (PC) (PY)

Sugars (g/L)

Fructose 14.41 ± 0.27c I I .98 ± 0.22b 4.33 ± 0.16a

Glucose 26.37 ± 0.47c 5.49 ± 0.17b 0.00 ± 0.00a

Sucrose 8.06 ± 0.06b 6.54 ± 0.33a 6.00 ± 0.23a

Mannose 7.69 ± 0.19b 2.16 ± 0.09a 1.35 ± 0.05a

Galactose 14.94 ± 0.20b 6.629 ± 0.19a 6.60 ± 0.19a

Arabinose 2.49 ± 0.30a 2.22 ± 0.13a 2.21 ± 0.20a

In total 74.98 ± 1.23c 35.06 ± 0.47b 20.49 ± 0.52a

Organic acids (g/L)

Citric acid 38.35 ± 0.13b 36.48 ± 0.06a 36.85 ± 0.04a a-Ketoglutaric acid (mg/L) 30.89 ± 2.40a 34.96 ± 0.96ab 36.45 ± 5.93b

Malic acid 0.97 ± 0.05c 0.33 ± 0.01b 0.25 ± 0.00a Succinic acid 1.83 ± 0.12a 2.18 ± 0.07b 3.17 ± 0.02c Lactic acid 0.09 ± 0.00a 0.71 ± 0.06b 1.13 ± 0.12c Acetic acid 0.04 ± 0.00a 0.52 ± 0.02b 0.48 ± 0.03b

Phenolic acids (mg/L)

Chlorogenic acids 120.86 ± 2.27b 99.57 ± 9.53a 66.11 ± 3.77ab Caffeic acid 469.88 ± 8.27a 340.83 ± 17.73b 219.57 ± 6.62b

Ferulic acid 18.64 ± 0.85a 16.83 ± 1.63a 11.96 ± 0.32a p-Coumaric acid 7.40 ± 0.21a 7.44 ± 0.017a 18.13 ± 0.43b

Alkaloids (mg/L)

Trigonelline 523.19 ± 15.71a 716.28 ± 18.57b 594.11 ± 12.62b

Theobromine 126.01 ± 3.33b I I I .95 ± 3.61a 99.89 ± 1.67ab

Theophylline 73.23 ± 2.17a 60.48 ± 1.94b 53.29 ± 1.01b

Caffeine 1386.29 ± 39.10a 1412.35 ± 21.69a 1395.78 ± 53.78a

Note: SCG: spent coffee grounds; PC: P. kluyveri FrootZen without yeast extract; PY: P. kluyveri FrootZen with 0.25% (w/v) yeast extracts a, b, c: Statistical analysis using ANOVA (n=3) at 95% confidence interval. Same letters indicate no significant difference between samples.

Table 7: Selected non-volatile compounds and their concentrations before and after fermentation with P. kluyveri FrootZen

Among the detected organic acids (citric acid, a-ketoglutaric acid, malic acid, succinic acid, lactic acid and acetic acid), succinic acid (2.18 g/L), lactic acid (0.71 g/L) and acetic acid (0.52 g/L) increased significantly, while citric acid and malic acid decreased significantly after fermentation. Moreover, the generation of succinic acid (3.17 g/L) and lactic acid (1.13 g/L) was further increased with addition of yeast extracts.

Phenolic acids and alkaloids were also quantified in Table 7. All detected phenolic acids declined (some significantly) after fermentation except for p-coumaric acid when yeast extracts was added. The supplementation of yeast extracts accelerated the generation of trigonelline and p-coumaric acid and accelerated the consumption of other detected phenolic compounds. Among the four alkaloids, trigonelline was produced significantly after fermentation while others decreased slightly, especially for significantly decreased caffeine when yeast extracts was added.

Changes of amino acids in the SCG hydrolysates fermented with P. kluyveri FrootZen were also measured (not shown). Eighteen kinds of amino acids were quantified. The addition of yeast extracts significantly enhanced the amount of amino acids in SCG hydrolysates. In general, a decreasing trend in the contents of most amino acids was observed. After fermentation, most amino acids except threonine, serine and cystine significantly decreased to low levels. Major utilization of the amino acids corresponded to the high levels of volatile compounds generated in the alcoholic beverage (see Table 8 below). The contents of threonine, serine and cystine increased after fermentation. The content of amino acids in the final SCG beverages ranged from 0-34.89 mg/L.

Volatile compounds

Volatile compounds quantified and measured are summarized in Table 8. In summary, there were acids (4), alcohols (2), esters (13), aldehydes (3), furan & ketones (4) and other odour-active substances (3). Among these compounds, 13 esters contribute to the pleasant and fruity aroma of SCG beverages which consist of ethyl acetate, ethyl hexanoate, ethyl octanoate, ethyl decanoate, ethyl dodecanoate, ethyl 2,4- hexadienoate, isoamyl acetate, propyl acetate, octyl acetate, benzyl acetate, 2- phenethyl acetate, 2-phenethyl propionate and furfuryl acetate. Moreover, the addition of yeast extracts significantly enhanced the generation of ethyl acetate, ethyl hexanoate, isoamyl acetate, and 2-phenethyl acetate.

In contrast with Example 3, two new esters (benzyl acetate, furfuryl acetate) were also detected in the beverage formed and the contents of some esters (e.g. ethyl acetate, isoamyl acetate and phenethyl acetate) were much higher than that in Example 3. Hence, the aroma characters of volatile compounds in Example 4 would be distinguished from that of other SCG related beverages formed in the other examples. Day 0 Day 14

Compound Identification methods SCG hydrolysate SCG beverage

FrootZen+

0% YE_ 0.25% YE FrootZen (PC) 0.25% YE (PY)

Acids

Pentanoic acid MS, LRI 1623 0.00 ± 0.00a 0.00 ± 0.00a 48.86± 4.41 b 73.40 ± 5.03c Hexanoic acid MS, LRI 1834 0.45 ± 0.05a 14.88 ± 1.76b 0.00 ± 0.00a 0.00 ± 0.00a

128.89 ±

Benzoic acid MS, LRI 2452 25.52c 36.98 ± 3.34b 0.00 ± 0.00a 0.00 ± 0.00a Octanoic acid MS, LRI 2038 0.00 ± 0.00a 0.00 ± 0.00a 30.84 ± 1.70b 93.64 ± 5.45c

Alcohols

1-Pentanol MS, LRI 1228 0.00 ± 0.00a 0.00 ± 0.00a 0.00 ± 0.00a 51 .06 ± 2.95b

2-Phenylethyl 34.27 ± 267.93 ± alcohol MS, LRI 1934 2.70a 40.94 ± 5.17a 12.26c 195.91 ± 11.17b

Esters

5850.99 ± 10482.89 ±

Ethyl acetate MS, LRI N.A. 0.00 ± 0.00a 0.00 ± 0.00a 337.23b 966.95c Ethyl hexanoate MS, LRI 1252 0.00 ± 0.00a 0.00 ± 0.00a 54.40 ± 6.82b 84.83 ± 6.53c Ethyl octanoate MS, LRI 1447 0.00 ± 0.00a 0.00 ± 0.00a 40.99 ± 5.54a 321.14 ± 41.91 b 125.66 ±

Ethyl decanoate MS, LRI 1653 0.00 ± 0.00a 0.00 ± 0.00a 11.01 b 292.22 ± 25.92c Ethyl dodecanoate MS, LRI 1829 0.00 ± 0.00a 0.00 ± 0.00a 0.00 ± 0.00a 50.11 ± 1.32b Ethyl 2,4- hexadienoate MS, LRI 1509 0.00 ± 0.00a 0.00 ± 0.00a 67.62 ± 5.12b 80.19 ± 1.90c

1713.15 ±

Isoamyl acetate MS, LRI 1147 0.00 ± 0.00a 0.00 ± 0.00a 121.23b 3387.50 ± 534.03c Propyl acetate MS, LRI N.A. 0.00 ± 0.00a 0.00 ± 0.00a 0.00 ± 0.00a 51 .07 ± 3.09b Octyl acetate MS, LRI 1477 0.00 ± 0.00a 0.00 ± 0.00a 18.88 ± 2.49b 21.19 ± 3.06b

Benzyl acetate MS, LRI 1744 0.00 ± 0.00a 0.00 ± 0.00a 48.48 ± 7.02b 50.48 ± 5.42b

2-Phenethyl 20470.10 ± 30988.75 ± acetate MS, LRI 1817 0.00 ± 0.00a 0.00 ± 0.00a 925.96b 1127.51b

2-Phenethyl propionate MS, LRI 1882 0.00 ± 0.00a 0.00 ± 0.00a 79.53 ± 6.20b 86.53 ± 5.12b

315.74 ±

Furfuryl acetate MS, LRI 1520 0.00 ± 0.00a 0.00 ± 0.00a 46.15b 286.55 ± 10.12b Aldehydes

127.92 ± 125.21 ±

Benzaldehyde MS, LRI 1540 21.65b 4.36b 40.19 ± 7.43a 120.71 ± 11.69b

Benzeneacetalde 141.31 ± hyde MS, LRI 1629 0.00 ± 0.00a 0.00 ± 0.00a 21.10b 401 .67 ± 20.10c 14.25 ±

Furfural MS, LRI 1488 2.09a 13.63 ± 2.65a 14.19 ± 1.67a 15.86 ± 0.54a

Furan & Ketones 2,5-Dimethylfuran MS, LRI 1033 0.00 ± 0.00a 0.00 ± 0.00a 43.17 ± 3.81 b 74.79 ± 4.27b

195.44 ± 216.44 ±

2-Pentylfuran MS, LRI 1247 15.12a 15.55a 0.00 ± 0.00b 0.00 ± 0.00b

2-Formyl-5- 602.72 ± 575.27 ± methylfuran MS, LRI 1574 59.27b 76.01 b 0.00 ± 0.00a 0.00 ± 0.00a

15.15 ±

2-Vinylfuran MS, LRI 2001 1.80a 11.72 ± 2.33a 13.21 ± 0.95a 17.26 ± 1.16a Phenolics

12.70 ± 10.23 ±

Guaiacol MS, LRI 1892 2.23c 1.20bc 5.67 ± 0.54ab 8.49 ± 0.56a

Others

99.54 ± 92.14 ±

2,4-Decadienal MS, LRI 1866 10.02b 10.29b 0.00 ± 0.00a 0.00 ± 0.00a

20.56 ±

3-Acetylpyrrole MS, LRI 1992 2.79ab 19.48 ± 1.55a 17.84 ± 1.16b 24.19 ± 0.51 a LRI: linear retention index; determined on a DB-FFAF column relative to C7-C40 hydrocarbons; YE: adding 0.25% yeast extract; Values are the mean of triplicate fermentations (± standard deviation); a,b,c Statistical analysis using ANOVA (n=3) at 95% confidence interval. PC: P. kluyveri FrootZen without yeast extract; PY: P. kluyveri FrootzenFrootZen with 0.25% (w/v) yeast extracts. Same letter indicates no significant difference between samples

Table 8: Selected volatile compounds and their concentrations ^g/L) before and after fermentation with P. kluyveri FrootZen

In this Example, the production of ethyl acetate, isoamyl acetate and 2-phenethyl acetate was significantly higher than other esters as seen in Figures 11A to C. Ethyl acetate presents an ethereal fruity smell when less than 10% in solution; isoamyl acetate gives a sweet fruity banana smell even at 1% solution; 2-phenethyl acetate contributes to a floral, rose like aroma, and the smell can last for 16 hours at 100%. Therefore, the high generation of ethyl acetate, isoamyl acetate and 2-phenethyl acetate could also been a safe and natural way to obtain condensed flavour and fragrance.

Examples 5, 6 and 7 - Formation of beverage from a non-Saccharomyces yeast and lactic acid bacteria

In these examples, beverages were formed by utilizing a non-Saccharomyces yeast (e.g. Lachancea thermotolerans Concerto) together with three different strains of a lactic acid bacterium (LAB), Oenococcus oeni to ferment the SCG hydrolysates with added yeast extracts to modulate flavour.

SCG hydrolysates prepared as described above. pH was adjusted to 5 by adding 10 M NaOH and subsequently, the SCG hydrolysates were pasteurized at 60°C for 30 min and cooled to room temperature before being fermented with L thermotolerans Concerto (Chr. Hansen, Horsholm, Denmark) and O. oeni (Chr. Hansen, Horsholm Denmark) with an initial cell count of approximately 5 log CFU/mL for L thermotolerans Concerto and approximately 6 log CFU/mL for O. oeni.

Three co-culture fermentations were conducted, including L thermotolerans Concerto and O. oeni Lalvin 31, L thermotolerans Concerto and O. oeni Enoferm Betaand L thermotolerans Concerto and O. oeni PN4. The hydrolysates have been defined as follows: Example 5 - Concerto + Lalvin; Example 6 - Concerto + Beta; and Example 7 - Concerto + PN4. The monoculture of L. thermotolerans Concerto (Concerto) was used as a control. The SCG hydrolysate fermentations were terminated after static incubation for 14 days at 20°C. Ethanol, sugars, organic acids, phenolics, alkaloids, amino acids and volatiles were analyzed.

The growth of yeast and O. oeni The growth of L thermotolerans Concerto under the four different combinations of LAB described above is shown in Figure 12A.

Yeast cell populations increased by 0.5 to 1.34 Log CFU/mL from day 0 to day 1 and decreased after day 1 in both control and co-culture fermentations. L thermotolerans Concerto slightly decreased when co-inoculated with O. oeni Lalvin 31, while the population of L. thermotolerans Concerto decreased sharply when co-inoculated with O. oeni PN4 and O. oeni Enoferm Beta from day 1 and no viable yeast cell could be detected by day 7.

The changes of the three O. oeni strains population showed similar trends as can be seen in Figure 12B. Cell growth increased gradually from day 0 to day 2 and decreased from day 2. The cell counts for O. oeni PN4 (initial 7.69 Log CFU/mL) and Enoferm Beta (initial 7.72 Log CFU/mL) increased to 9.41 Log CFU/mL and 9.30 Log CFU/mL at day 2, respectively. The population of O. oeni Lalvin 31 (initial 6.26 Log CFU/mL) increased to 8.09 Log CFU/mL at day 2. The lower initial biomass of O. oeni Lalvin 31 was ascribed to its slow growth in pre-culture. The increments in cell counts were approximately 1.7 Log CFU/mL for the three oenococcal strains.

Ethanol content

As seen in Figure 13, the ethanol content in the SCG hydrolysates fermented with the co-culture of L thermotolerans Concerto and O. oeni Lalvin 31 (Example 5), co-culture of L. thermotolerans Concerto and O. oeni Enoferm Beta (Example 6), and co-culture of L thermotolerans Concerto and O. oeni PN4 (Example 7) were 1.55%, 0.58%, and 0.58%, respectively, which were significantly lower than that (4.94%, v/v) of L. thermotolerans Concerto monoculture fermentation.

Non-volatile components Non-volatile compounds such as sugars, organic acids, phenolic acids, alkaloids, and amino acids were measured. Sugars including fructose, glucose, sucrose, mannose, galactose and arabinose were detected in the SCG hydrolysates. The results are as shown in Table 9.

Day o Fermented SCG (Day 14)

Compound SCG Concerto Concerto Concerto hydrolysates concerto + La Ivin + Beta + PN4

Sugars (g/L)

Fructose 18.70 ± 0.62c 3.92 ± 0.14b 2.64 ± 0.63a 2.18 ± 0.19a 2.22 ± 0.12a Glucose 52.68 ± 2.12b 3.85 ± 0.29a 4.15 ± 0.04a 6.90 ± 0.92a 6.95 ± 0.13a

Sucrose 10.83± 0.31c 2.48 ± 0.08a 4.76 ± 0.07b 4.55 ± 0.38b 4.94 ± 0.28b

Mannose 16.27 ± 0.74c 4.05 ± 0.08a 4.54 ± 0.25a 7.11 ± 0.82b 7.16 ± 0.12b

Galactose 16.88 ± 0.49c 4.40 ± 0.50a 6.65 ± 0.28b 7.13 ± 0.71 b 6.88 ± 0.39b

Arabinose 2.59 ± 0.16a 2.49 ± 0.10a 2.60 ± 0.19a 2.54 ± 0.06a 2.51 ± 0.07a

Total 117.96 ± 2.99c 21.19 ± 0.77a 25.34 ± 1.33a 30.42 ± 2.96b 30.65 ± 0.77b

Organic acids (g/L)

Citric acid 35.05 ± 0.47b 33.73 ± 0.12b 27.32 ± 1.08a 32.54 ± 0.15b 33.45 ± 0.07b a-Ketoglutaric acid

(mg/L) 34.55 ± 4.14c 9.74 ± 0.09a 20.32 ± 0.71 b 19.58 ± 1.04b 14.19 ± 0.32a

Malic acid 0.38 ± 0.02c 0.04 ± 0.00a 0.17 ± 0.01 b 0.36 ± 0.02c 0.34 ± 0.01c Pyruvic acid 0.01 ± 0.00a 0.25 ± 0.01e 0.16 ± 0.01d 0.04 ± 0.00b 0.06 ± 0.00c Succinic acid 2.67 ± 0.04a 3.75 ± 0.17a 14.86 ± 0.63b 16.36 ± 2.01 b 14.34 ± 0.70b Lactic acid 0.12 ± 0.00a 0.53 ± 0.02a 18.46 ± 0.16b 20.24 ± 2.04bc 22.37 ± 1.10c

Acetic acid 0.14 ± 0.00a 0.45 ± 0.01b 5.04 ± 0.19d 4.67 ± 0.09c 5.16 ± 0.05d

Phenolic acids (mg/L)

Chlorogenic acid 137.35 ± 4.80c 45.011 ± 5.64a 56.78 ± 4.46a 78.80 ± 5.59b 81.86 ± 5.74b

596.96 ± 649.66 ± 640.01 ±

Caffeic acid 28.69b 27.75c 11 86bc 55.07 ± 5.89a 73.25 ± 2.03a Ferulic acid 12.44 ± 0.14a 25.21 ± 0.89b 24.94 ± 2.63b 24.88 ± 2.39b 29.40 ± 1.58b p-Coumaric acid 5.14 ± 0.05a 10.95 ± 0.34d 8.27 ± 0.23c 6.68 ± 0.77b 6.72 ± 0.18b

Alkaloids (mg/L)

507.23 ± 471.31 ± 506.18 ± 511.27 ±

Trigonelline 10.19b 441.84 ± 7.20a 1.40ab 27.70b 23.55b

Theobromine 131.18 ± 7.64b 26.26 ± 2.08a 34.30 ± 1.84a 33.67 ± 1.46a 26.72 ± 7.84a

Theophylline 68.83 ± 2.13b 26.40 ± 1.53a 47.86 ± 5.72a 36.71 ± 5.61 a 38.44 ± 1.85a 1199.99 ± 1158.66 ± 1273.93 ± 1032.26 ± 1004.66 ±

Caffeine 37.21 ab 36.11ab 185.42b 71 70ab 48.79a

Note: SCG: spent coffee grounds; Concerto: monoculture of L thermotolerans Concerto; Concerto+Lalvin: Co-culture of L. thermotolerans Concerto and O. oeni Lalvin 31 ; Concerto+Beta: Co-culture of L. thermotolerans Concerto and O. oeni Enoferm Beta; Concerto+PN4: Co-culture of L. thermotolerans Concerto and O. oeni PN4. a, b, c: Statistical analysis using ANOVA (n=3) at 95% confidence interval. Same letters indicate no significant difference between samples

Table 9: Selected non-volatile compounds and their concentrations before and after fermentation with L. thermotolerans Concerto and O. oeni Non-volatile components in Example 5 (Concerto+Lalvin)

In Example 5 (Concerto + Lalvin), the total sugar content declined significantly from 117.96 g/L to around 25.34 g/L after fermentation. All sugars were significantly consumed by yeast and/or bacteria during fermentation except for arabinose. The total sugar content (25.34 g/L) remaining in the fermented SCG hydrolysates in Example 5 was similar to that of monoculture fermentation of L thermotolerans Concerto.

Organic acids such as citric acid, a-ketoglutaric acid, malic acid, succinic acid, lactic acid and acetic acid were detected. Succinic acid (14.86 g/L), lactic acid (18.46 g/L), acetic acid (5.04 g/L) and pyruvic acid (0.16 g/L) increased significantly, while citric acid, a-Ketoglutaric acid and malic acid decreased significantly after fermentation. Moreover, succinic acid and lactic acid produced in the co-culture of L thermotolerans Concerto with O. oeni Lalvin 31 were significantly higher than those in the monoculture of L. thermotolerans Concerto.

Phenolic acids and alkaloids were also quantified in Table 9. After fermentation, ferulic acid and p-coumaric acid increased significantly; chlorogenic acids decreased significantly, while caffeic acid kept stable. Among the four alkaloids, theobromine and theophylline decreased significantly while others kept stable after fermentation.

Amino acids especially threonine, serine and tyrosine were markedly consumed by L thermotolerans Concerto and O. oeni Lalvin 31 as compared with the consumption of amino acids in the monoculture fermentation of L thermotolerans Concerto. In general, a decreasing trend for most amino acids was observed except for glycine. The remaining content of glycine was higher before fermentation. The contents of amino acids in the final SCG beverages ranged from 1.20-78.75 mg/L after fermentation.

Non-volatile compounds in Example 6 (Concerto+Beta) In Example 6 (Concerto + Beta), the total sugar contents declined significantly from 117.96 g/L to around 30.42 g/L after fermentation, which was higher than that (21.19 g/L) remained monoculture fermentation of L thermotolerans Concerto. All sugars were significantly consumed by yeast and/or bacteria during fermentation except for arabinose. Organic acids including citric acid, a-ketoglutaric acid, malic acid, succinic acid, lactic acid and acetic acid were detected. Succinic acid (16.36 g/L), lactic acid (20.24 g/L), acetic acid (4.67 g/L) and pyruvic acid (0.04 g/L) increased significantly; a-Ketoglutaric acid decreased significantly while malic acid kept stable before and after fermentation. Moreover, the contents of succinic acid and lactic acid produced in the co-culture of L thermotolerans Concerto with O. oeni Enoferm Beta were significantly higher than those generated in the monoculture of L thermotolerans Concerto.

Phenolic acids and alkaloids in Example 6 were also quantified as shown in Table 9. For phenolic acids, p-coumaric acid and ferulic acid increased significantly, chlorogenic acids and caffeic acid decreased significantly.

Among the four alkaloids, theobromine and theophylline decreased significantly, while trigonelline and caffeine kept stable when taking SCG hydrolysates before fermentation as a reference. The amount of trigonelline was significantly produced as compared with the monoculture of L thermotolerans Concerto. Most amino acids were consumed by L. thermotolerans Concerto and O. oeni Enoferm Beta during fermentation. Among the detected amino acids, serine and alanine were markedly consumed by L thermotolerans Concerto and O. oeni Enoferm beta. In general, a decreasing trend for most amino acids was observed. Interestingly, the remaining contents of phenylalanine, lysine and arginine markedly were significantly higher than those remained in the monoculture of L thermotolerans Concerto after fermentation. The contents of amino acids in the final SCG beverages ranged from 3.43-90.39 mg/L.

Non-volatile compounds in Example 7 (Concerto+PN4)

In Example 7 (Concerto + PN4), the total sugar contents declined significantly from 117.96 g/L to around 30.65 g/L after fermentation, which was higher than that (21.19 g/L) of monoculture fermentation of L. thermotolerans Concerto. All sugars were significantly consumed by yeast and/or bacteria during fermentation except for arabinose.

Among all detected organic acids, succinic acid (14.34 g/L), lactic acid (22.37 g/L), acetic acid (5.16 g/L) and pyruvic acid (0.06 g/L) increased significantly; a-Ketoglutaric acid decreased significantly, while malic acid and citric acid kept stable before and after fermentation.

Phenolic acids and alkaloids in Example 7 were quantified in Table 9. For phenolic acids, p-coumaric acid increased significantly, chlorogenic acids, caffeic acid and ferulic acid decreased significantly.

Among the four alkaloids, theophylline and theophylline decreased significantly after fermentation. Meanwhile, trigonelline decreased significantly as compared with that of the monoculture fermentation of L thermotolerans Concerto.

Most amino acids were consumed by L thermotolerans Concerto and O. oeni PN4 during fermentation. Among the detected amino acids, serine and tyrosine were markedly consumed by L. thermotolerans Concerto and O. oeni PN4. In general, a declining trend for most amino acids was observed compared with SCG hydrolysates before fermentation. Interestingly, the contents of amino acids such as valine, Leucine, phenylalanine and arginine after fermentation were markedly higher than those remained in the monoculture fermentation of L thermotolerans Concerto. The contents of amino acids in the final SCG beverages ranged from 2.67-88.54 mg/L.

Volatile compounds

Volatile compounds play a significant role on the aroma and flavour of fermented SCG beverages such as methyl acetate (fragrant, fruity), methyl lactate (strawberry flavour), furfural (almond fragrant), acetoin (buttery odor), diacetyl (buttery odor). The differences between the kinds and concentrations of volatile compounds could contribute to the different characteristics for the fermented SCG beverages.

The identified volatile compounds of SCG hydrolysates before and after fermentation in Examples 5, 6 and 7 are shown in Table 10. In particular, volatile acids (13), alcohols (6), esters (13), aldehydes (4), ketones (6), furans (3), phenols (6), terpenoids (3), pyrazines (3) and pyrroles (3) were detected.

In Example 5 (Concerto + Lalvin), there was a higher content of methyl acetate, methyl lactate, furfural, acetoin, and diacetyl in the coculture fermented SCG hydrolysates when compared with that of the monoculture fermentation of L. thermotolerans Concerto. In Example 6 (Concerto + Beta), the contents of 2-furanmethanol, methyl acetate, methyl lactate, furfural, acetoin, diacetyl, and volatile phenols (e.g. p-cresol) were significantly higher than those in the single culture fermentation of L thermotolerans Concerto. In Example 7 (Concerto + PN4), the fermented SCG hydrolysates showed significantly higher contents of 2-furanmethanol, methyl acetate, methyl lactate, furfural, diacetyl, 4- ethylguaiacol, 2,5-dimethylfuran and pyrrole-2-carboxaldehyde compared with those in the single culture fermentation of L thermotolerans Concerto.

In summary, the prototypes of SCG beverages of Examples 5, 6 and 7 were different from each other in the contents of sugars, organic acids, phenolic acids, and volatile compounds as discussed above. Moreover, large amounts of organic acids (e.g. succinic acid and lactic acid) were generated from SCG fermentation.

Whilst the foregoing description has described exemplary embodiments, it will be understood by those skilled in the technology concerned that many variations may be made without departing from the present invention.

Day 0 Fermented SCG hydrolysates (Day 14)

Compounds Identification methods Unfermented

CC CL CB CP

SCG hydrolysates

Acids a-Pyrone-6-carboxylic acid MS, LRI 1364 0.00 ± 0.00a 72.39 ± 0.54c 21.53 ± 0.32b 1.23 ± 0.01 a 0.98 ± 0.00a

Propanoic acid MS, LRI 1542 20.48 ± 0.11d 4.11 ± 0.04a 4.67 ± 0.05a 14.64 ± 0.21 c 10.90 ± 0.09b

2-Propenoic acid MS, LRI 1642 5.26 ± 0.04a 3.60 ± 0.32a 2.43 ± 0.20a 11.30 ± 0.12b 2.54 ± 0.00a Butanoic acid MS, LRI 1630 30.19 ± 0.12d 5.36 ± 0.03b 7.54 ± 0.11b 2.52 ± 0.03a 19.03 ± 0.09c Pentanoic acid MS, LRI 1631 4.72 ± 0.03e 2.89 ± 0.01d 0.50 ± 0.00a 1.56 ± 0.02b 2.23 ± 0.03c

3-Methylcrotonic acid MS, LRI 1803 8.72 ± 0.03c 3.84 ± 0.02a 3.92 ± 0.04a 7.53 ± 0.04b 7.01 ± 0.03b Hexanoic acid MS, LRI 1848 15.81 ± 0.09b 7.37 ± 0.09a 9.45 ± 0.08a 17.14 ± 0.11b 21.26 ± 0.12c (£)-3-Hexenoic acid MS, LRI 1922 0.00 ± 0.00a 8.06 ± 0.09c 3.58 ± 0.01b 3.04 ± 0.03b 3.70 ± 0.01 b Octanoic acid MS, LRI 2063 1.50 ± 0.01 a 2.76 ± 0.03a 6.53 ± 0.07b 19.23 ± 0.07d 16.81 ± 0.04c Nonanoic acid MS, LRI 2169 0.87 ± 0.01 b 1.75 ± 0.00c 0.26 ± 0.00a 0.99 ± 0.01 b 0.93 ± 0.00b Decanoic acid MS, LRI 2276 0.00 ± 0.00a 0.81 ± 0.01b 1.79 ± 0.03c 2.11 ± O.OOcd 2.42 ± 0.02d Benzoic acid MS, LRI 2454 3.71 ± 0.05ab 6.03 ± 0.03c 2.96 ± 0.03a 6.21 ± 0.03c 4.27 ± 0.04b Benzeneacetic acid MS, LRI 2578 1.24 ± 0.02b 0.82 ± 0.01a 0.89 ± 0.02ab 1.16 ± 0.01 ab 0.94 ± 0.01 ab Alcohols

Isoamyl alcohol MS, LRI 1219 1.19 ± 0.01 a 240.72 ± 1.84d 91.54 ± 1.00c 61 .36 ± 0.40b 45.55 ± 1.82b

2-Heptanol MS, LRI 1323 57.31 ± 0.32d 5.04 ± 0.05a 6.21 ± 0.03a 25.93 ± 0.02c 17.48 ± 0.11 b

2,3,3-Trimethyl-2- butanol MS, LRI 1394 1.98 ± 0.02a 5.20 ± 0.06b 6.21 ± 0.04b 13.17 ± 0.02c 36.98 ± 0.09d

Furfuryl MS, LRI 1677 4.11 ± 0.02a 32.02 ± 0.39b 30.84 ± 0.46b 77.02 ± 0.13d 68.88 ± 0.27c

Benzyl alcohol MS, LRI 1910 2.44 ± 0.01 a 4.66 ± 0.02c 3.58 ± 0.03b 4.07 ± 0.02b 5.34 ± 0.03d

2-Phenylethyl alcohol MS, LRI 1952 0.00 ± 0.00a 647.87 ± 108.50 ±

3.12d 154.76 ± 1.60c 0.23b 97.64 ± 0.71 b

Aldehydes

2-Propenal MS, LRI 1120 3.37 ± 0.03a 93.99 ± 1.14b 1.21 ± 0.01a 0.00 ± 0.00a 5.85 ± 0.04a

Furfural MS, LRI 1477 1.24 ± 0.12a 11.59 ± 0.09ab 20.97 ± 0.13b 88.17 ± 0.85c 93.92 ± 0.19c

Benzaldehyde MS, LRI 1538 126.56 ± 0.99b 29.44 ± 0.38a 22.53 ± 0.16a 22.25 ± 0.08a 26.67 ± 0.22a

2.4-dimethyl MS, LRI 1835 10.18 ± 0.03d 5.54 ± 0.03a 5.43 ± 0.03a 7.44 ± 0.03b 9.06 ± 0.05c benzaldehyde

Esters

Ethyl acetate MS, LRI / 0.00 ± 0.00a 94.27 ± 0.81 b 318.452 ± 3.49c 69.82 ± 0.80b 80.49 ± 0.89b Ethyl hexanoate MS, LRI 1226 0.00 ± 0.00a 4.11 ± 0.05b 2.21 ± 0.03b 19.87 ± 0.16d 16.81 ± 0.10c Ethyl 2,4-hexadienoate MS, LRI 1512 0.00 ± 0.00a 5.98 ± 0.01d 0.68 ± 0.01c 0.67 ± 0.00c 0.27 ± 0.00b Ethyl octanoate MS, LRI 1431 0.00 ± 0.00a 19.95 ± 0.25c 7.42 ± 0.03b 4.64 ± 0.01 b 5.59 ± 0.07b Ethyl nonanoate MS, LRI 1534 0.00 ± 0.00a 2.72 ± 0.00c 0.54 ± 0.00b 0.00 ± 0.00a 0.00 ± 0.00a Ethyl decan oate MS, LRI 1637 0.00 ± 0.00a 17.12 ± 1 87d 10.45 ± 0.67c 0.55 ± 0.05a 6.87 ± 0.14b Ethyl benzoate MS, LRI 1627 0.00 ± 0.00a 0.00 ± 0.00a 0.92 ± 0.00d 0.26 ± 0.00b 0.49 ± 0.01 c Isoamyl acetate MS, LRI 1117 0.00 ± 0.00a 9.94 ± 0.06c 8.02 ± 0.15c 3.51 ± 0.02b 2.17 ± 0.01 b Methyl acetate MS, LRI 1313 0.00 ± 0.00a 0.00 ± 0.00a 29.42 ± 0.07d 13.41 ± 0.09c 11.12 ± 0.10b Methyl 2-furoate MS, LRI 1589 0.00 ± 0.00a 5.12 ± 0.03b 5.00 ± 0.06b 9.42 ± 0.07c 8.87 ± 0.08c Methyl lactate MS, LRI 1324 0.00 ± 0.00a 7.26 ± 0.08b 44.67 ± 0.00c 60.05 ± 0.63d 82.87 ± 1.02e 2-furanmethyl acetate MS, LRI 1242 0.00 ± 0.00a 1.97 ± 0.03b 3.86 ± 0.04c 7.38 ± 0.08d 8.33 ± 0.09d 2-Phenethyl acetate MS, LRI 1828 0.00 ± 0.00a 3.01 ± 0.04cd 3.50 ± 0.03d 2.21 ± 0.02b 2.63 ± 0.03bc Furans

2.5-Dimethylfuran MS, LRI / 22.53 ± 0.01 ab 25.36 ± 20.14 ±

0.13ab 0.15ab 62.65± 13.72b 77.98 ± 4.83b 2-Acetylfuran MS, LRI 1518 36.51 ± 0.47b 15.56 ± 0.26a 15.64 ± 0.11 a 32.83 ± 0.24b 30.92 ± 0.10b 2-Acetyl-5-methylfuran MS, LRI 1629 14.02 ± 0.09c 6.58 ± 0.02a 6.5 ± 0.03a 11.30 ± 0.12b 11.10 ± 0.07b Ketones 2-Heptanone MS, LRI 1205 2.63 ± 0.02a 0.00 ± 0.00a 16.64 ± 0.23b 44.31 ± 0.17d 35.31 ± 0.19c Diacetyl MS, LRI 1750 0.00 ± 0.00a 0.00 ± 0.00a 1.49 ± 0.02a 3.31 ± 0.01 c 2.50 ± 0.03b

101.49 ±

Acetoin MS, LRI 1301 0.00 ± 0.00a 7.34 ± 0.09a 197.95 ± 1.45c 76.69 ± 1.75b 1.06b

2-Nonanone MS, LRI 1387 3.96 ± 0.01a 0.00 ± 0.00a 45.52 ± 0.40b 52.35 ± 0.35b 88.93 ± 0.99c

2.5-Hexanedione MS, LRI 1516 0.00 ± 0.00a 0.00 ± 0.00a 2.35 ± 0.01a 26.37 ± 0.24c 22.02 ± 0.12b Methyl cyclopentenolone MS, LRI 1842 4.13 ± 0.19b 0.82 ± 0.14a 2.43 ± 0.11ab 3.32 ± 0.01 ab 4.02 ± 0.04b

Pyrazines

2.6-Dimethylpyrazine MS, LRI 1337 5.40 ± 0.03d 2.00 ± 0.02a 2.91 ± 0.03b 3.98 ± 0.02c 3.82 ± 0.05c 2-Ethyl-6- methylpyrazine MS, LRI 1396 7.76 ± 0.07b 2.63 ± 0.04a 2.41 ± 0.02a 2.78 ± 0.02a 3.07 ± 0.02a 2-Ethyl-3,5- dimethylpyrazine MS, LRI 1449 6.36 ± 0.01 c 2.92 ± 0.01 b 2.99 ± 0.05b 1.37 ± 0.02a 1.44 ± 0.01 a Pyrroles 2-Acetylpyrrole MS, LRI 1989 14.47 ± 0.14c 9.79 ± 0.03ab 8.22 ± 0.05a 12.33 ± 0.08bc 12.84 ± 0.16c Pyrrole-2- carboxaldehyde MS, LRI 2048 21 .98 ± 0.16c 8.65 ± 0.07a 10.15 ± 0.03a 19.50 ± 0.01b 19.08 ± 0.11 b

1 -Methyl-2-formylpyrrole MS, LRI 2127 3.98 ± 0.01d 2.21 ± 0.00b 1.54 ± 0.01a 3.45 ± 0.01 cd 3.23 ± 0.05c

Terpenoids frans-Linalool oxide MS, LRI 1444 15.65 ± 0.08c 3.40 ± 0.03a 1.98 ± 0.01a 8.75 ± 0.11 b 9.83 ± 0.04b

Linalool MS, LRI 1546 5.03 ± 0.05a 3.40 ± 0.03a 4.06 ± 0.04a 20.31 ± 0.09b 19.72 ± 0.18b

Terpineol MS, LRI 1655 1.17 ± 0.01 b 0.69 ± 0.01a 1.06 ± 0.01ab 2.04 ± 0.02c 2.54 ± 0.02d

Volatile phenols m-Cresol MS, LRI 1145 3.17 ± 0.01 a 21.50 ± 0.02bc 17.44 ± 0.12b 34.79 ± 0.28d 26.05 ± 0.23c

Guaiacol MS, LRI 1876 6.52 ± 0.07b 3.51 ± 0.02a 3.67 ± 0.02a 4.44 ± 0.03a 5.90 ± 0.03b

Phenol MS, LRI 2020 11.31 ± 0.07c 8.10 ± 0.06b 6.10 ± 0.02a 9.80 ± 0.08b 9.32 ± 0.09b

4-Ethylguaiacol MS, LRI 2047 1.78 ± 0.01a 3.80 ± 0.04b 5.67 ± 0.02c 15.58 ± 0.10d 15.88 ± 0.05d p-Cresol MS, LRI 2097 2.21 ± 0.02c 1 .17 ± 0.01 a 0.81 ± 0.01a 1.92 ± 0.02bc 1.68 ± 0.02b

LRI: linear retention index; determined on a DB-FFAF column relative to C7-C40 hydrocarbons; Concerto: monoculture of L. thermotolerans Concerto; Concerto+Lalvin: Co-culture of L. thermotolerans Concerto and O. oeni Lalvin 31 ; Concerto+Beta: Co-culture of L. thermotolerans Concerto and O. oeni Enoferm Beta; Concerto+PN4: Co-culture of L. thermotolerans Concerto and O. oeni PN4; Values are the mean of triplicate fermentations (± standard deviation); a,b,c,d,e Statistical analysis using ANOVA (n=3) at 95% confidence interval. Same letter indicates no significant difference between samples

5 Table 10: Selected volatile compounds and their concentrations ^g/L) before and after fermentation with L. thermotolerans and O. oeni