TITLE: MATE BEVERAGE

FIELD OF THE INVENTION The present invention relates to a process for preparing a mate extract comprising caffeine, theobromine, theophylline and antioxidant compounds. The mate extract can be used as an ingredient in the preparation of a mate syrup or mate beverage base formulation for making mate beverages, such as carbonated mate beverages.

BACKGROUND OF THE INVENTION

Beverages, such as teas, containing caffeine are consumed throughout the world. The extraction of tea material is known in the art. For example, US 5,427,806 discloses a process for the production of green tea extracts using a gelatin solution to remove oxidized or polymerized flavanols. However, this process uses an acidified aqueous solution containing preservatives to obtain the green tea extract from the green tea materials. A later patent by the same group, US 5,780,086 discloses color and shelf stable beverage compositions containing tea extracts including a beverage syrup for use in juices, carbonated and non-carbonated beverages. However, the tea product claimed contains green tea extracts obtained by contacting the green tea materials with an acidified solution containing preservatives, which is subsequently gelatin treated, clarified and concentrated. In addition, this patent also contemplates the addition of synthetic compounds, such as caffeine, to the beverage compositions. A tea extract and process is also disclosed in United States statutory invention registration number H1 ,628. The process there describes extraction of tea materials with water, mixing the extract containing solution with protein, acidifying the protein containing extract and separating the precipitate. One of the preferred proteins listed is gelatin of any particle size derived from animal protein. Furthermore, compositions including the use of synthetic compounds such as caffeine are described. A recently published US patent application 20050186314,

discloses the use of silica or silica-containing compounds to remove proteins and polysaccharides from green tea extracts in beverages that are sold hot. The application describes extraction of green tea leaves with hot water prior to silica treatment and contemplates the addition of acid after the extraction procedure.

Recent US patent applications have been published describing the use of Ilex species in formulations, as either the main component or part of the formulation, for human therapeutic purposes. The genus Ilex is found worldwide in subtropical and tropical regions. /. paraguariensis is known as mate and is the most commercialized plant of South America. US patent applications 20050118293 and 20050089591 describe compositions derived from Ilex species, particularly Ilex paraguariensis, having lowered caffeine and tannin concentrations, as oral delivery formulations for use in the treatment of human physiological and medical conditions. However, the application claims an extraction process from native mate plant material using supercritical carbon dioxide solutions under pressure. The application also describes mate compositions having predetermined characteristics, such as a lowered amount of caffeine, elevated amounts of caffeoyls, and/or lowered amounts of tannin compounds compared to the native mate plant materials. However, the specification notes that some of the chemical constituents found in mate, such as caffeine, theobromine and theophylline may be purchased from a chemical supply company. In US patent application 20050089584, a method and composition for oral delivery of Areca and mate or theobromine for use in the treatment of mental and physical conditions is disclosed. In US patent application 20050037025, a method and compositions comprising kava and mate or theobromine for the treatment of conditions related to mental and physical fatigue is claimed. Extraction methods disclosed in both these applications include steam distillation, liquid extraction (using dicholoromethane or petroleum ether), hydroalcoholic extraction, Soxhlet extraction and supercritical carbon dioxide solution under pressure. The specification from both of these patent applications also notes that

theobromine may be obtained from mate, other sources or made by synthetic means known to those skilled in the art.

Prior efforts to make a mate syrup for use in mate beverages are disclosed in German patent number 1106154 issued in 1959. The German patented process employs an aqueous extraction of mate leaf using boiling water under pressure. The tea brew extract is then treated with preservatives such as sodium benzoate (0.08%) and a high concentration of citric acid (6.5%). The extract is then stored for 14 days, treated with a silica compound and filtered through an asbestos filter. However, the product from the patented German process is not certified organic, relies on a high degree of acidity and on the addition of chemical preservatives to achieve a functional shelf-life. In addition, caffeine levels in the German product are achieved through the addition of synthetic caffeine, rather than utilizing caffeine derived solely from the mate plant. Therefore there is a need in the art for a process of preparing a mate extract rich in natural caffeine, theobromine, theophylline and antioxidant compounds completely derived from mate plant materials, without the addition of preservatives and synthetic materials/compounds for use in the preparation of a preservative-free mate beverage base formulation.

SUMMARY OF THE INVENTION

The present application relates to a specialized and unique formulation and production procedure for a preservative-free liquid mate extract rich in natural caffeine, theobromine, theophylline and antioxidant compounds. The application provides a process for preparing a clarified mate extract comprising the steps of:

(a) providing plant material from a plant of the species //ex;

(b) producing a raw extract from the plant material which comprises caffeine, theobromine and theophylline; (c) adding gelatin to the raw extract;

(d) adding silica to the gelatin-treated solution of step (c) and allowing the mixture to settle;

(e) freezing the silica, gelatin containing extract of step (d); and (T) thawing the extract of step (e) and separating the clarified liquid supernatant from the solids to obtain a clarified mate extract and an extract cake. A significant quantity of natural mate caffeine remains bound in the extract cake of step (T). In one aspect, the extract cake of step (T) is subjected to an additional step where the extract cake is washed with an aqueous phase, preferably cold water in a preferred embodiment. In this additional step, the aqueous solution added to the extract cake is separated from the solids to obtain a mate cake wash.

The clarified mate extract can be used as an ingredient for preparation of a mate syrup or mate beverage base formulation for use in mate beverages including carbonated mate beverages. The final product is advantageous in that it complies with organic certification regulations and delivers caffeine and other phyto-stimulants without the addition of synthetic materials or preservatives.

In one embodiment, the invention provides a mate beverage base formulation comprising the following ingredients:

(a) clarified mate extract; (b) water;

(c) sugar;

(d) colour; and

(e) acid.

In a specific embodiment, the water component of the mate beverage base formulation is the mate cake wash.

In another embodiment, the invention provides a process for preparing the mate beverage base formulation comprising the steps of:

(a) providing a clarified mate extract;

(b) adding water to the extract; (c) agitating and heating the mixture of steps (a) and (b) until all ingredients are in solution; and (d) adding sugar, acid and colour.

In another embodiment, the preparation of a mate beverage base formulation may be accomplished using the mate cake wash as the water component.

In another embodiment, the invention provides for mate beverages, including carbonated mate beverages, prepared using the mate beverage base formulation of the invention.

Other features and advantages of the present invention will become apparent from the following detailed description. It should be understood, however, that the detailed description and the specific examples while indicating preferred embodiments of the invention are given by way of illustration only, since various changes and modifications within the spirit and scope of the invention will become apparent to those skilled in the art from this detailed description.

DETAILED DESCRIPTION OF THE INVENTION

As noted previously, the present invention relates to a clarified mate extract that can be used for the preparation of a mate syrup or mate beverage base formulation. Accordingly, the present invention provides:

(a) providing plant material from a plant of the species Ilex; (b) producing a raw extract from the plant material which comprises caffeine, theobromine and theophylline;

(c) adding gelatin to the raw extract;

(d) adding silica to the gelatin-treated solution of step (c) and allowing the mixture to settle; (e) freezing the silica, gelatin containing extract of step (d); and

(f) thawing the extract of step (e) and separating the clarified liquid supernatant from the solids to obtain a clarified mate extract and an extract cake.

As noted above, a significant quantity of natural mate caffeine remains bound in the extract cake of step (f). In one aspect, the extract cake of step

(T) is subjected to an additional step where the extract cake is washed with an aqueous phase, preferably cold water in a preferred embodiment. In this

additional step, the aqueous solution added to the extract cake is separated from the solids to obtain a mate cake wash.

Plant Material The plant material is obtained from a plant of the Ilex species.

Preferably, the plant material is obtained from Ilex paraguariensis. The leaves and stems of the plant are toasted, preferably in wood - fueled toasting ovens at 88°C to 92°C, preferably at 90 ⁰ C for 13 to 17 minutes, preferably for 15 minutes. Toasted plant materials are milled with a hammer mill to a particle size of about 2 to 10mm, preferably to a particle size of 3 mm to 5 mm. Toasted materials are sifted to separate leaf and stem materials. Separated plant parts are re-combined to obtain a mixture of about 93% to 97% leaf and 3% to 7% stem w/w, preferably 95% leaf and 5% stem w/w.

Green Mate Extract Concentrate

The green mate extract concentrate may be produced from green mate leaves. Leaf material may be extracted with hot water at about 9O ⁰ C to 100 ⁰ C, preferably 100 ⁰ C, under agitation at 40 to 60 rpm, preferably 60 rpm. The resulting liquid is concentrated under vacuum and contains a minimum of 8% to 10% caffeine, preferably 10% caffeine, 0.7% to 1.0% theobromine, preferably 1.0% theobromine, and 0% to 0.2% theophylline, preferably 0.2% theophylline, derived from the mate plant.

In one aspect, the toasted organic mate leaf material and green mate extract concentrate is the plant material of step (a).

Extraction

The extraction process of step (b) is carried out using an extraction vessel, holding tanks, transfer pipes and related equipment constructed of food-grade stainless steel. A person skilled in the art would readily be able to select appropriate extraction vessels. In a particular aspect, the extraction of step (b) is carried out using an extraction vessel that is totally enclosed with a top loading port and a screened bottom discharge valve to facilitate drainage

of the finished extract. In another particular aspect, the aqueous mate extraction of step (b) is carried out in a double-jacketed kettle.

In the mate extraction step, the extraction vessel is at 30 psi and is filled with the required amount of water heated to 9O ⁰ C to 100 ⁰ C, preferably heated to 100 ⁰ C. In one aspect, a circular agitator is employed to facilitate optimal contact of the mate plant material with the extraction solvent. Once the extraction vessel is filled with water, heated to the desired temperature, and the agitator is running, the Ilex plant material is added. In one aspect, the Ilex plant material added is the toasted organic mate leaf material, followed by the green mate extract concentrate. In another aspect, the Ilex plant material is the green mate extract concentrate, followed by the toasted organic mate leaf material. The extraction mix of step (b) is maintained at the desired temperature under agitation ("thermal extraction interval"). In one aspect, the extraction mix of step (b) is agitated for 8 to 12 minutes. In another aspect, the extraction mix of step (b) is agitated for 10 minutes. In one aspect, the extraction mix of step (b) is agitated at a speed of 40 to 60 rpm. In another aspect, the extraction mix of step (b) is agitated at a speed of 60 rpm. In a particular aspect, the extraction mix of step (b) is maintained at 100 ⁰ C, and agitated at 60 rpm for 10 minutes. Once the required thermal extraction interval is achieved for extraction, the agitator is stopped. The liquid fraction ("hydro-thermal extract") is drained, cooled, and strained into a sterilized holding tank. In one aspect, the holding tank is stainless steel. In one aspect, the hydro-thermal extract is cooled using a heat exchanger immediately following the end of the thermal extraction interval. In another aspect, the hydro-thermal extract is cooled from the extraction temperature in step (b) to 5°C to 10°C. In a particular aspect, the hydro-thermal extract is cooled to 5°C. In one aspect, the hydro-thermal extract is strained through a screen into the sterilized stainless steel holding tank. In a particular aspect, the hydro-thermal extract is strained through a 250 μM screen into a sterilized stainless steel holding tank.

Immediately following completion of the hydro-thermal extraction phase, the remaining mate solid materials ("mate mash") are transferred from the

extraction vessel and pressed. Pressing of the mate mash should be completed quickly in order to maintain a minimum mate mash temperature of 30°C to 5O ⁰ C which facilitates full release of essential oils and other flavour- rich compounds. In a particular aspect, a minimum mate mash temperature of 30 ⁰ C is maintained during pressing. The mate mash is pressed under a selected peak pressure for a period of 5 to 10 minutes, preferably 10 minutes, or until the flow of liquid stops.

In one aspect, the mate mash is pressed using a rack and cloth pack press. In another aspect, the press must be capable of achieving and maintaining a peak pressure. In a particular aspect, the press must be capable of achieving and maintaining a peak pressure of 250 km/cm ² . A person skilled in the art would readily be able to select an appropriate press. Accessories required for pressure extraction using the rack and cloth pack press include High Density Polyethylene (HDPE) frame separators, HDPE press frames, and synthetic press cloth with an opening size of 0.5 to 1.5 mm, preferably with an opening size of 1 mm. A person skilled in the art would readily be able to select other appropriate food-safe frame separators and press frames.

The liquid is collected and strained through a screen. In one aspect, the collected liquid is strained through a 250 μM screen. The strained liquid is then combined with the hydro-thermal extract in the sterilized stainless steel holding tank prior to the clarification step.

Clarification The raw mate extract from step (b) is clarified using a synergistic two- part colloidal clarification process. In the first step, the raw mate extract is treated with gelatin and silica to initiate floe formation. In the second step, a secondary floe formation and sedimentation reaction is catalyzed through a post-seeding freeze treatment ("cryfloculation"). The inventors have demonstrated that gelatin and silica treatments must be used in combination and in addition to the subsequent freeze treatment step, in order to achieve improved clarification. The inventors have shown that only gelatin-silica

treatments or only freeze treatment alone do not achieve desired clarification as when both steps are applied together and in sequential order.

Briefly, clarification is commenced by first contacting the raw mate extract with a gelatin solution (step (c)), allowing the solution to mix, subsequently adding a silica suspension (step (d)), and further allowing the solution to mix. Prior to cryfloculation (step (e)), the seeded mixture is chilled and allowed to settle in a tank without any disturbance to allow for sedimentation of solids. After this period, the supernatant liquid is drawn off and frozen to initiate the cryfloculation reaction. The gelatin treatment is applied to the raw mate extract at 0.10 % w/w to

0.15% w/w of the total extract formula, preferably at 0.15% w/w of the total extract formula. The prepared silica suspension is then added immediately following the gelatin treatment. Silica treatment is applied at 0.10% w/w to 0.15% w/w of the total extract formula, preferably at 0.15% w/w of the total extract formula.

The specific combination of gelatin, followed by silica results in synergistic clarification of the raw mate extract. This is due to the fact that the gelatin applied is specially formulated to act in combination with a matched silica gel suspension; this specific interaction results in the initiation of floe seeding or floe formation.

Once the gelatin solution and silica suspension have been added and blended, the floe seeded raw mate extract is chilled to 3°C to 6°C, preferably chilled to 5 ⁰ C and allowed to settle in a tank without any physical disturbance for a period of 44 to 48 hours, preferably for 48 hours. Following the chill period, the supernatant liquid is drawn off from the sedimented solids and is chilled to -15°C to -20 ⁰ C, preferably to -18°C for at least 20 hours, preferably for at least 24 hours, or until extract is solidly frozen throughout. Once full extract solidification is achieved, the frozen extract is allowed to thaw with no physical disturbance (step (f)). Once the frozen extract is fully thawed, the clarified supernatant liquid is separated from the sediment and is strained. In one aspect, the clarified supernatant liquid is siphoned. In another aspect, the siphoned clarified supernatant is strained

through a 200 μM to 250 μM screen. In a particular aspect, the siphoned supernatant is strained through a screen that is 250 μM. The siphoned and strained supernatant is the clarified mate extract. The remaining sediment is the "extract cake". A significant quantity of natural mate caffeine remains bound in the extract cake of step (f). In one aspect, the extract cake of step (f) is subjected to an additional step where the extract cake is washed with an aqueous phase, preferably cold water in a preferred embodiment. In this additional step, the aqueous solution added to the extract cake is separated from the solids to obtain a mate cake wash. The mate cake wash contains re-captured caffeine and other bound xanthines, such as theobromine and theophylline. One part extract cake is combined with 5.5 parts cold potable water at 15 ⁰ C to 2O ⁰ C, preferably at 2O ⁰ C, in a vessel. In one aspect, the vessel is stainless steel. This mixture is then agitated for 8 to 12 minutes, preferably for 10 minutes, at a speed of 40 rpm to 60 rpm, preferably at 60 rpm. Following this step, the mixture is filtered. A person skilled in the art would readily be able to select appropriate filter aids. The filtered product is the mate cake wash. In one aspect, the mate cake wash is used as the water component in the preparation of a mate beverage base formulation.

Process for Preparation of Mate beverage base formulation

The clarified mate extract can be used for the preparation for the mate syrup or mate beverage base formulation. In one embodiment, the invention provides a mate beverage base formulation comprising the following ingredients:

(a) clarified mate extract;

(b) water;

(c) sugar;

(d) colour; and (e) acid.

In a preferred embodiment, the clarified mate extract is combined with water and is heated under agitation to a temperature of 6O ⁰ C to 7O ⁰ C,

preferably to 70 ⁰ C. In another preferred embodiment, the mate cake wash is used as the water component that is combined with the clarified mate extract. The sugar, acid and colour are added and mixed until all ingredients are completely in solution. The acid is added to adjust the final acidity of the solution. In one aspect, the sugar added is NOP organic cane. In another aspect, the colour added is organic aromatic caramel colour. Acids that may be added to the solution include acetic acid, tartaric acid, malic acid, fumaric acid, lactic acid, phosphoric acid and citric acid. Preferably, the acid added is phosphoric acid or citric acid. Most preferably, the acid added is citric acid. Phosphoric acid may be added in the range of 0.9% - 4.5%. Citric acid may be added in the range of 1 % - 2%. In a preferred embodiment, the prepared mate beverage base formulation contains 10% - 30% clarified mate extract, preferably 18% - 22% clarified mate extract, 50% - 65% NOP organic cane sugar, preferably 56% - 60% NOP organic cane sugar, 1% - 10% organic caramel colour, preferably 2% - 5% organic caramel colour, 10% - 25% water, preferably 15% - 18% water and 1 % - 2% citric acid, preferably 1.2% - 1.4% citric acid. In a specific embodiment, the prepared mate beverage base formulation contains 20.03% clarified mate extract, 58.4% NOP organic cane sugar, 3.56% organic caramel colour, 16.76% water and 1.25% citric acid (to achieve a final acidity of 1.4 g/100g w/w measured as citric acid). The beverage may also contain other suitable ingredients including organic lime juice and/or organic lemon oil. Once ingredients are mixed, the brix is verified and the finished mate syrup or mate beverage base formulation is filled in suitable containers. In one aspect, the brix is 65. In another aspect, plastic lined drums, or plastic pails are suitable containers. In another aspect, aseptic packing in sterile bag-in box containers are suitable containers. The final beverage preferably comprises about 0.15 to about 0.30 mg/mL of caffeine, more preferably about 0.18 to about 0.22 mg/mL. In a 300 ml_ serving, there will preferably be about 55 to 65 mg of caffeine. The mate syrup or mate beverage base formulation may be stored at

5°C if used within 7 days, otherwise the mate syrup or mate beverage base formulation is stored frozen.

Mate Beverages Prepared Using Mate Beverage Base Formulation

The mate syrup or mate beverage base formulation is used to produce a mate beverage, such as a ready-to-drink, sparkling Yerba Mate beverage. In one aspect, a carbonated mate beverage is prepared by combining the mate beverage base formulation with water in a holding tank. Preferably, the mate beverage is 65 brix and the water is filtered. The holding tank is preferably a sterile stainless steel tank. The mixture is agitated to a uniform mix (single strength beverage mix). Brix and acid parameters of the mixture are measured and adjusted if required. The single strength mixture is then filtered into a sterile stainless steel holding tank. A person skilled in the art would readily be able to select appropriate filter aids. The filtered mixture is carbonated with food-grade carbon dioxide and the mate beverage is filled into retail packaging. In a preferred aspect, the filtered mixture is carbonated to a level of 3.0 volumes with food-grade carbon dioxide. In another preferred aspect, glass bottles are used as retail packaging. In a preferred embodiment, the prepared sparkling yerba mate beverage contains 5% - 20% mate beverage base formulation, preferably 10% - 12% mate beverage base formulation and 80% - 95% carbonated water, preferably 86% - 90% carbonated water. In a specific embodiment, the prepared sparkling yerba mate beverage contains 11.23% mate beverage base formulation and 88.77% carbonated water.

The following non-limiting examples are illustrative of the present invention: EXAMPLES

Example 1 1.0 Summary

This Example outlines a specialized and unique formulation and production procedure for a certified organic, preservative-free liquid mate extract rich in natural caffeine, theobromine and antioxidant compounds. The procedure entails an aqueous extraction of a combination of toasted mate (Ilex paraguahensis) leaves and a mate leaf extract concentrate. Water

extraction is followed by extract clarification using synergistic colloidal clarification and cryo-floculation methods. The final clarified extract is then used as an ingredient for preparation of a 65 brix mate syrup for use in carbonated mate beverages. The final product is unique in that it complies with organic certification regulations and delivers caffeine and other phyto- stimulants without the addition of synthetic materials.

2.0 EXTRACTION

2.1 RAW MATERIALS

2.1.1 Toasted Organic Mate Leaf

The plant material required for extract production consists of a mixture of toasted leaves and stems of certified organic, NOP (USDA National Organic Program, 7 CFR Part 205) compliant Ilex paraguariensis Saint- Hillaire, commonly known as Yerba Mate or Paraguay Tea. Leaf and stem materials are toasted in wood - fueled toasting ovens at 90 ⁰ C for 15 minutes. Toasted plant materials are milled with a hammer mill to a particle size of 3 mm to 5 mm. Toasted materials are sifted to separate leaf and stem materials. Separated plant parts are re-combined to obtain a mixture of 95% leaf and 5% stem w/w. Finished plant material must be packed and stored at maximum 18% moisture to avoid microbial proliferation.

2.1.2 Green Mate Extract Concentrate The mate extract concentrate required for the mate preparation consists of a certified organic extract of the green leaves of certified organic, NOP compliant Ilex paraguariensis Saint Hillaire plant material. The green mate leaf material is extracted with hot water at 100°C, under agitation at 60 rpm. The resulting liquid is concentrated under vacuum and must contain a minimum of 8.0% caffeine and 0.7% theobromine derived from the mate plant.

2.1.3 Water

Potable water is used as the extraction solvent and must conform to Canadian Drinking Water Standards and the following water quality characteristics:

pH: 6.8- 7.8

Hardness: < 50 mg/L

E. coli: 0 cfu/100 ml_

Coliforms: 0 cfu/100 mL

Total chlorine: 0 mg/L

2.2 EQUIPMENT

2.2.1 Design and Construction

The extraction vessel, holding tanks, transfer pipes and related equipment should be constructed of food-grade stainless steel.

2.2.2 Extraction Vessel

The mate extraction is carried out in a double-jacketed kettle at 30 psi heated with over-critical hot water. A circular agitator with a speed of 60 rpm is required to facilitate optimal contact of the mate plant material with the extraction liquid. The extraction vessel must be totally enclosed with a top loading port and a screened bottom discharge valve to facilitate drainage of the finished extract.

2.2.3 Heat Exchanger

A heat exchanger is required to cool the primary extract from 100 ⁰ C to 5°C to10°C immediately following the end of the thermal extraction interval.

2.2.4 Rack and Cloth Pack Press A rack and cloth pack press is required for pressure extraction of the mate mash following hydro-thermal extraction. The press must be capable of achieving and maintaining a peak pressure of 250 km/cm ² . Accessories

required for pressure extraction include HDPE frame separators, HDPE press frames, and synthetic press cloth with an opening size of 1 mm.

2.3 EXTRACTION PROCEDURE The following steps must be followed in order to achieve an effective extraction and subsequent clarification reactions.

2.3.1 Hydro-Thermal Extraction

The extraction vessel is filled with the required amount of water which is heated to 100 ⁰ C. At 100 ⁰ C with the agitator running, the mate leaf is added, followed by the mate extract. The extraction mix is maintained at 100 ⁰ C under agitation for 10 minutes. Once the required extraction interval is achieved, the agitator is stopped and the liquid fraction is drained off, cooled to 5°C to 10 ⁰ C, and strained through a 250 μm screen into a sterilized stainless steel holding tank.

2.3.2 Pressure Extraction of Solids

Immediately following the completion of the hydro-thermal extraction phase, the remaining solids are transferred from the extraction vessel to a rack and frame pack press. This must be completed as quickly as possible in order to maintain a minimum mash temperature of 30°C. The mash is pressed to a pressure of 250 km/cm ² which is held for 10 minutes, or until the flow of liquid stops. The collected liquid is passed through a 250 μm screen and is combined with the hydro-thermal extract. The combined liquids constitute the raw extract.

3.0 CLARIFICATION

3.1. RAW MATERIALS

3.1.1 GELATIN

Dry, powdered gelatin is used as one of the ingredients of a synergistic two-part colloidal clarification process. The gelatin must be a specifically formulated, positively charged material specifically formulated for the reduction of polyphenols and tannins by acting in combination with a silica gel suspension. The Bloom Value of the gelatin must be in the range of 90-100.

Gelatin treatment is applied at 0.15% w/w of the total extract formula. The gelatin treatment is added prior to the silica treatment.

The gelatin is applied as a gelatin solution prepared as follows. Dry gelatin powder is blended with 5 parts cold potable water under constant agitation. The gelatin suspension is blended for a period of 20 minutes and is subsequently diluted with a further 4 - 5 parts of hot potable water to bring the solution to 40 ⁰ C - 50 ⁰ C. The finished gelatin solution should be used as soon as possible.

3.1.2 SILICA

A specialized Silica suspension is used in combination with a matched gelatin to initiate floe formation. The silica suspension must consist of a 30% active, negatively charged, colloidal solution of highly purified silica.

Silica treatment is applied at 0.15% w/w of the total extract formula. The silica treatment is added immediately following gelatin treatment.

3.2. CLARIFICATION PROCESS

The raw mate extract is treated with silica and gelatin to initiate floe formation. Floe formation and sedimentation is catalyzed through a post- seeding freeze treatment

3.2.1 FLOC SEEDING

The raw mate extract is treated as follows at a temperature range of 5°C to 10°C: a) With the raw extract under gentle agitation, add the prepared gelatin solution and allow to mix for 2 to 3 minutes, b) Under continued agitation, add the silica sol and continue

agitation for 2 to 3 minutes. The raw mate extract is now seeded for floe formation. c) Allow the treated extract to stand undisturbed at 5 ⁰ C (or between 3 ⁰ C to 6 ⁰ C) for 48 hours to allow for sedimentation of formed solids.

3.2.2 CRYFLOCULATION

In order to achieve full floe formation and sedimentation of solids, a freezing interval is required immediately following the initial floe seeding step.

Once gelatin and silica solutions have been allowed to react for 48 hours, the treated raw mate extract must be separated from formed sediment and chilled to -18°C for at least 24 hrs. The extract must be fully frozen to achieve the required effect.

Once full extract solidification has been achieved, the extract is allowed to thaw with no physical disturbance. Once the extract is fully thawed, the clarified supernatant liquid is siphoned off and strained through a 250 μm screen. This becomes the "clarified mate extract". The remaining sediment is referred to as "extract cake".

3.2.3 EXTRACT CAKE WASH A significant quantity of natural mate caffeine remains bound in the extract cake. In order to re-capture this caffeine and other bound xanthines, the extract cake is washed with cold water as follows:

Combine 1 part extract cake with 5.5 parts cold potable water in a stainless steel mixing vessel. Agitate mixture for 30 minutes at 20 ⁰ C (60 rpm mixer speed). Add 15% w/w Diatomaceous Earth filter aid (water permeability

2 darcies) and filter through a DE filter press. Finished, filtered product is the

"cake wash".

The cake wash is used as the aqueous ingredient in the mate base formula.

4.0 MATE BEVERAGE BASE FORMULATION

The mate beverage base formulation is the commercial mate preparation which will be commercially available for the production of a ready- to-drink, sparkling Yerba Mate beverage.

4.1 FORMULATION

The mate beverage base formulation consists of clarified mate extract, NOP organic cane sugar, organic aromatic caramel colour, water and citric acid. Key quality parameters are as follows:

Brix: 65. 0 pH: 2.6

Acidity: 1.4 g/100 g w/w as citric acid

4.2 PREPARATION The clarified mate extract is combined with the water component and is heated under agitation to 70 ⁰ C. Cane sugar, citric acid and colour are added and mixed until all ingredients are completely in solution. The ingredients are added so that the mate beverage base formulation contains 20.03% clarified mate extract, 58.4% cane sugar, 3.56% colour, 16.76% water and 1.25% citric acid (to achieve a final acidity of 1.4 g/100g w/w measured as citric acid). Verify brix and fill finished syrup in suitable containers. Plastic lined drums, or plastic pails are suitable. Store at 5°C if syrup is used within 7 days, otherwise store frozen. An alternative is aseptic packing in sterile bag-in box containers.

5.0 QUALITY CONTROL AND STANDARDIZATION

The finished mate beverage base formulation must be analyzed for levels of naturally occurring caffeine, theobromine, theophylline and antioxidant capacity (ORAC). High Performance Liquid Chromatography (HPLC) is used for analysis of caffeine, theobromine, theophylline. A microplate fluorescence assay is used to determine the ORAC value (Oxygen Radical Absorbance Capacity). The mate beverage base is formulated to

deliver 55 mg - 65 mg of naturally occurring caffeine per 300 mL serving with an ORAC value of 6,000 - 7,000 μmol TE/L.

6.0 PREPARATION METHOD FOR CARBONATED MATE BEVERAGE The 65 brix mate beverage base formulation and filtered water (0.20 μm) are combined into a sterile stainless steel tank and are mechanically agitated to a uniform mix (single strength beverage mix). Brix and acid parameters are measured and adjusted if required. The single strength mixture is then filtered through a diatomaceous earth filter followed by a 1.0 μm pad filter and a 0.45 μm membrane filter into a sterile stainless steel holding tank. The filtered mixture is carbonated to a level of 3.0 volumes with food-grade carbon dioxide and is filled into retail packaging (glass bottles).

While the present invention has been described with reference to what are presently considered to be the preferred examples, it is to be understood that the invention is not limited to the disclosed examples. To the contrary, the invention is intended to cover various modifications and equivalent arrangements included within the spirit and scope of the appended claims.

All publications, patents and patent applications are herein incorporated by reference in their entirety to the same extent as if each individual publication, patent or patent application was specifically and individually indicated to be incorporated by reference in its entirety.