Title of Invention

Fermented tea infusions

This application claims priority from GB2016419.0 filed on 16 October 2020, the contents and elements of which are herein incorporated by reference for all purposes.

Field of the Invention

The present invention relates to a fermented infusion of a tea leaf, and in particular to beverages comprising the fermented infusion. The fermented infusions described herein exhibit low levels of acetic acid (e.g. about 1 % (w/w) or less) and low levels of alcohol (e.g. about 1.2% (v/v) or less).

Background

Fermentation is used in the beverage industry in order to develop and/or diversify the flavours of the drinks produced using plant materials.

For example, alcoholic beverages such as wine and beer are typically produced using yeasts to carry out alcohol fermentation of grapes and cereals, respectively. In these processes, the yeast is added to the plant material in the presence of a carbohydrate source such as sugar and fermentation converts the carbohydrate source into ethanol and carbon dioxide. Fermentation has also been carried out on infusions (e.g. teas) made from plant materials in order to develop and/or diversify their flavours.

There is a demand for a low alcohol or alcohol-free beverage without sacrificing the flavours consumers’ associate with the “adult taste” of fermented beverages such as wine and beer.

Labelling requirements for low-alcohol and alcohol-free beverages vary between countries. In the UK, a low-alcohol beverage has an alcohol strength of between 0.05 and 1.2% alcohol by volume (ABV) and an alcohol-free beverage has an alcohol strength of less than 0.05% ABV. Other countries classify alcohol- free beverages as having an alcohol strength of less than 0.5% ABV.

Kombucha (also known as tea mushroom, tea fungus, or Manchurian mushroom when referring to the culture) is a fermented, slightly alcoholic, lightly effervescent, sweetened black or green tea drink commonly intended as a functional beverage for its perceived health benefits. Kombucha is produced by fermenting infusions of plant material (e.g. infusions of a tea leaf) using a symbiotic culture of bacteria and yeast (i.e. the “SCOBY”). The microbial populations in a SCOBY vary: the yeast component generally includes Saccharomyces cerevisiae, along with other species, while the bacterial component almost always includes the acetic acid bacteria Gluconacetobacter xylinus to oxidize yeast-produced alcohols to acetic acid (and other acids). Kombucha fermentation typically takes place over a week or longer. The SCOBY culture is a symbiotic growth of acetic acid bacteria and osmophilic yeast species in a zoogleal mat or biofilm. The living bacteria are said to be probiotic, one of the reasons for the drink's popularity.

However, kombucha contains acetic acid (i.e. vinegar), which is polarising to the consumer in terms of a sour taste, especially in an infusion-based beverage such as a tea beverage. This limits the appeal of kombucha.

EP0791296B1 discloses a process for making a fermented beverage and a fermented beverage obtained by this process. Example 2 of EP0791296B1 discloses a process where a fermented infusion of green tea is produced. According to this example, 7% sucrose is added to a green tea infusion, which is then adjusted to a pH of 5 and fermented using a Saccharomyces cerevisiae yeast and a Gluconobacter suboxydans bacteria. Fermentation takes place at 30 °C for 7 hours under aerobic conditions and the resulting fermented tea is filtered and pasteurised. EP0791296B1 describes the resulting sweet fermented tea as having an apple taste, a pH of 4.47, an ethanol content of 0.05% and an acetic acid content of 0.032%.

While the process disclosed in Example 2 of EP0791296B1 reportedly resulted in a sweet fermented tea having a low alcohol and a low acetic acid content, it was recognised that the infusion was fermented with the S. cerevisiae yeast and G. suboxydans bacteria for a relatively short time (7 hours) compared to other processes for making fermented teas involving similar yeast and bacteria. It was hypothesized that the short period of fermentation would mean that only a limited amount of fermentation was actually taking place and that this would account for the low levels of acetic acid and ethanol produced.

To test this hypothesis, the present inventors attempted to reproduce Example 2 of EP0791296B1 and measure how much of the initial carbohydrate feedstock (sucrose) was used during the fermentation reaction. The results are reported in Example 3 below and demonstrate that most of the initial sugars are retained in the sweet fermented tea produced. The sweet fermented tea was also described by tasters as having characteristics such as "sugary, thick" and “really sweet”. These results suggest that there is very little fermentation taking place in the prior art process and that the resulting sweet fermented tea produced contains a high sugar content. The limited fermentation taking place in the prior art process is expected to limit the development and/or diversification of flavours in the fermented infusions and would lack sensory properties associated with fermented infusions (e.g. fermented flavours, aromas and/or aftertastes).

The present invention has been devised in light of the above considerations.

Summary of the Invention

The present inventors made the surprising discovery that it was possible to produce fermented infusions of a tea leaf (e.g. a green tea leaf) that overcame the limitations of EP0791296B1. In particular, it was shown that by selecting particular microorganisms (e.g. yeasts) to carry out the fermentation process that were capable of efficiently consuming the carbohydrate feedstock, fermented infusions could be produced that exhibited low levels of acetic acid and low levels of alcohol, whilst also exhibiting a modulated sensory profile caused by fermentation of plant materials (see Figure 1).

Accordingly, in one aspect provided herein is a fermented infusion of a tea leaf, wherein the fermented infusion has: an acetic acid content of about 1 % (w/w) or less; an ethanol content of about 1.2% (v/v) or less; and a carbohydrate content of about 60 g/L or less, and/or a Brix value of about 6 ⁰ Brix or less.

In some embodiments, the fermented infusion has a carbohydrate content of about 50 g/L or less, and/or a Brix value of about 5 ° Brix or less. In some embodiments, the fermented infusion has a carbohydrate content of about 40 g/L or less, and/or a Brix value of about 4 ° Brix or less. In some embodiments, the fermented infusion has a carbohydrate content of about 3 g/L or less, and/or a Brix value of about 3 ° Brix or less.

In some embodiments, the fermented infusion has a carbohydrate content of between about 10 g/L and 60 g/L and/or a Brix value of between about 1 ° and 6 ° Brix. In some embodiments, the fermented infusion has a carbohydrate content of between about 20 g/L and 50 g/L and/or a Brix value of between about 2 ° and 5 ° Brix. In some embodiments, the fermented infusion has a carbohydrate content of between about 20 g/L and 40 g/L and/or a Brix value of between about 2 ° and 4 ° Brix. In some embodiments, the fermented infusion has a carbohydrate content of between about 20 g/L and 40 g/L and/or a Brix value of between about 2 ° and 3 ° Brix. In some embodiments, the fermented infusion has an acetic acid content of about 0.8% (w/w) or less. In some embodiments, the fermented infusion has an acetic acid content of about 0.5% (w/w) or less. In some embodiments, the fermented infusion has an acetic acid content of about 0.3% (w/w) or less. In some embodiments, the fermented infusion has an acetic acid content of less than 0.3% (w/w).

In some embodiments, the fermented infusion has an alcohol content of about 1% (v/v) or less. In some embodiments, the fermented infusion has an alcohol content of about 0.75% (v/v) or less. In some embodiments, the fermented infusion has an alcohol content of about 0.5% (v/v) or less.

In some embodiments, the fermented infusion has a pH of about 4 or less.

In yet another aspect provided herein is a fermented infusion of a tea leaf, wherein the fermented infusion has an acetic acid content of 1.5% (w/w) or less, an ethanol content of about 1.2% (v/v) or less; and a carbohydrate content of about 60 g/L or less, and/or a Brix value of about 6 ° Brix or less.

In some embodiments, the fermented infusion has an acetic acid content of 1.4 % (w/w) or less. In some embodiments, the fermented infusion has an acetic acid content of 1.3 % (w/w) or less. In some embodiments, the fermented infusion has an acetic acid content of 1.2 % (w/w) or less. In some embodiments, the fermented infusion has an acetic acid content of less than 1.1% (w/w).

In some embodiments, the fermented infusion has an alcohol content of about 1% (v/v) or less. In some embodiments, the fermented infusion has an alcohol content of about 0.75% (v/v) or less. In some embodiments, the fermented infusion has an alcohol content of about 0.5% (v/v) or less.

In some embodiments, the fermented infusion has a pH of about 4 or less.

The fermented infusions of a tea leaf described herein may be defined as having a modulated (e.g. improved) sensory profile as a result of the fermentation (i.e. when compared to an unfermented infusion of the tea leaf). For example, the fermented infusion of the tea leaf may have one or more sensory characteristics selected from a list consisting of appearance, aroma, flavour, mouthfeel, and aftertaste modified and/or improved, when compared to an unfermented infusion of the tea leaf. The fermented infusion may have a more fermented aroma, a more fermented flavour, and/or a more fermented aftertaste, when compared to an unfermented infusion of the tea leaf. The fermented infusion may having a less tea flavour, a less tea aftertaste, a less bitter flavour and/or a less bitter aftertaste, when compared to an unfermented infusion of the tea leaf. In some embodiments, the fermented infusion of the tea leaf is produced by:

(a) preparing an infusion of the plant material (e.g. tea leaf); and

(b) adding a fermentation microorganism to the infusion.

In some embodiments, the fermented infusion of the tea leaf is produced by:

(a) providing an infusion of the plant material (e.g. tea leaf); and

(b) adding a fermentation microorganism to the infusion.

The microorganism used to ferment the infusion (“the fermentation microorganism”) may comprise one or more yeasts, for example a yeast of the family Saccharomycetaceae. In preferred cases, the microorganism used for fermentation comprises or consists of a Saccharomyces cerevisiae (S. cerevisiae) yeast.

Yeast such as S. cerevisiae are well known microorganisms that can be used for fermentation of plant materials. S. cerevisiae is commonly associated with the efficient conversion of carbohydrates into ethanol and carbon dioxide during fermentation. Such yeasts are referred to as “winemakers” yeasts and are commercially available from various sources, including from commercial entities such as WeissBioTech GmbH, Max Baldinger AG, Brouwland and local wine making suppliers. While these winemaker yeasts reduces the initial carbohydrate feedstock present at the start of the fermentation reaction and advantageously modulate the sensory profile of the plant material (e.g. producing a more fermented aroma, flavour and/or aftertaste), these yeasts are often associated with the corresponding production of high amounts of alcohol.

The inventors determined that it was possible to utilise S. cerevisiae winemaker yeasts in fermentation processes that benefits from the highly efficient conversion of carbohydrates associated with these yeasts but without the significant production of ethanol. Accordingly, these yeasts could be used to produce fermented infusions with modulated sensory profile that exhibit the desired low alcohol, low acetic acid, low sugar properties. One way of doing this is to screen known S. cerevisiae winemaker yeasts for their ability to remove initial sugars and produce ethanol and acetic acid. A person of average skill in the art would be able to obtain S. cerevisiae winemaker yeasts (e.g. from the commercial sources identified above), carry out the screening assay disclosed herein on these yeasts and, by utilising the disclosed selection criteria, select a yeast to produce fermented beverages having the desired properties described herein. For example, the yeast (e.g. a S. cerevisiae yeast) may be capable of producing a test fermented green tea infusion with a pH of about 4 or less, less than about 7 g/L ethanol, less than about 0.5 g/L acetic acid, and less than about 5 g/L total sugar in a fermentation screening assay, the fermentation screening assay comprising preparing a test green tea infusion by contacting 10 g of green tea leaf with water at 80 °C for 45 minutes; adding 20 g/L sucrose or glucose to 5 ml of the test green tea infusion; adding 0.2 - 0.4 g/L of the yeast to the test green tea infusion; allowing the test green tea infusion to ferment at 28 °C for 6 days to produce the test fermented green tea infusion.

In some embodiments, producing the fermented infusion does not involve the use of Gluconacetobacter xylinus In other words, in some embodiments the fermentation microorganism(s) added to the infusion of the plant material does not comprise Gluconacetobacter xylinus. In some embodiments, producing the fermented infusion does not involve using an acetic acid bacteria. In other words, in some embodiments the fermentation microorganism(s) added to the infusion of the plant material does not comprise an acetic acid bacteria. Acetic acid bacteria are bacteria within the family Acetobacteraceae and comprise those bacteria of the genera Acetobacter, Acidomonas, Ameyamaea, Asaia, Gluconacetobacter,

Gluconobacter, Granulibacter, Kozakia, Neoasaia, Neokomagataea, Saccharibacter, Swaminathania and Tanticharoenia.

In another aspect, the present invention provides a process for preparing a fermented infusion of a plant material comprising a tea leaf, the process comprising preparing an infusion of the plant material, then adding a fermentation microorganism to the infusion. The fermented infusion may have an acetic acid content of about 1% (w/w) or less, an ethanol content of about 1.2% (v/v) or less, and a carbohydrate content of about 60 g/L or less (and/or a Brix value of about 6 ° Brix or less).

For example, the process may comprise the steps of:

(a) preparing an infusion of a plant material comprising a tea leaf (i.e. tea),

(b) (optionally) adding a carbohydrate feedstock, preferably a sugar, to the infusion;

(c) (optionally) filtering the infusion to remove remaining solid material of the plant material;

(d) adding a fermentation microorganism to the infusion;

(e) fermenting the infusion under conditions suitable to the microorganism to produce the fermented infusion; and

(f) (optionally) filtering the infusion to remove the microorganism. The fermentation microorganism may be as further described herein. For example, it may comprise or consist of a S. cerevisiae yeast, and/or may have been identified using the screening assay described herein.

The carbohydrate content in the fermented infusion will vary depending on the total amount of carbohydrate feedstock added at the start of fermentation step (c). Since the fermentation microorganism uses the carbohydrate feedstock to carry out fermentation, the carbohydrate content in the fermented infusion will always be reduced (e.g. at least 10% reduced, at least 20% reduced, or at least 30% reduced) compared to the amount of carbohydrate added at the start of fermentation step (c).

Typically, the total amount of carbohydrate added at the start of fermentation step (c) is about 60 g/L or less, or more preferably about 50 g/L or less. This is different to processes involving the production of sweet fermented teas such as in EP0791296B1 , which discloses a process where 7% sucrose (i.e. 70 g/L) is added to the infusion prior to fermentation taking place.

In some embodiments, the total amount of carbohydrate added at the start of fermentation step (c) is from 30 - 50 g/L, and/or wherein the Brix value of the infusion at the start of fermentation step (c) is 3-5 ° Brix. In some embodiments, the infusion is fermented until the carbohydrate content in the infusion is from 20 - 25 g/L, and/or wherein the infusion is fermented until the Brix value of the fermented infusion is 2-2.5 ° Brix. In some embodiments the infusion is fermented for between 1 and 5 days, e.g. between 2 and 3 days.

Also provided by the present invention is a fermented infusion obtainable using a process as described herein. Also provided is a fermented infusion obtained using a process as described herein.

Also provided is a beverage comprising the fermented infusion described herein. The beverage will also be characterised by having the low levels of acetic acid (e.g. about 1% (w/w) or less) and low levels of alcohol (e.g. about 1.2% (v/v) or less) described herein in the context of the fermented infusions.

In some embodiments, the beverage includes ingredients in addition to the fermented infusion such as any one or more of those selected from the group consisting of: a flavouring agent (e.g. a natural flavouring agent), a stabiliser, a preservative, an extract (e.g. fruit, malt, roots, ginger and/or acai), a sugar (e.g. sucrose), a pH modulating agent, a fruit juice, a sweetener (e.g. stevia), a colouring agent (e.g. a natural colouring agent), a vitamin (e.g. a B vitamin), a mineral (e.g. magnesium), and an amino acid (e.g. L-theanine). Accordingly, the process disclosed herein may include a step of adding one or more of these additional ingredients to the fermented infusion to produce a beverage. These ingredients can be added before, during or after the fermentation step, but will most typically be added after the fermentation step. The process disclosed herein may also include a step of adding a liquid (e.g. water) to the fermented infusion, i.e. to dilute the fermented infusion.

In some embodiments, at least one ingredient selected from the group consisting of citric acid, diammonium phosphate, sodium hydrogen carbonate, sodium hydroxide, malic acid, lactic acid, phosphoric acid, disodium citrate, trisodium citrate, potassium hydroxide and potassium carbonate is added as a pH modulating agent before, during or after the fermentation step. In some embodiments citric acid and/or diammonium phosphate is added as a pH modulating agent before the fermentation step.

In some embodiments, the beverage has a carbohydrate (e.g. sugar) content of between about 20 g/L and about 60 g/L, and/or a Brix value of between about 2 ° Brix and 6 ⁰ Brix; or the beverage comprises a sweetener in an amount that, together with any sugar present in the beverage, provides a sweetness in the beverage equivalent to between about 2 ° Brix and 6 ⁰ Brix.

Further details of suitable beverages are described herein.

The invention includes the combination of the aspects and preferred features described except where such a combination is clearly impermissible or expressly avoided.

Brief Description of Drawings

Embodiments and experiments illustrating the principles of the invention will now be discussed with reference to the accompanying figures in which:

Figure 1. Sensory profile of a fermented green tea infusion produced according to the process described (“fermented base”). Results are compared to an unfermented green tea infusion (“unfermented base infusion”). Modalities assessed include appearance (Ap), Aroma (Ar), Flavour (F), Mouthfeel (Mf) and Aftertaste (At). Modalities that exhibit a statistically significant difference at 95% confidence are boxed. NS: Not significant at 5%, *: significant at 5%, **: significant at 1 %, ***: significant at 0.1 %.

Figure 2. Sensory profile of a fermented green tea infusion produced according to the process in Example 5. Results are compared among the fermented green teas fermented with an S. cerevisiae yeast strain HF13011, HF13004 or HF13005, respectively. Modalities assessed include appearance (Ap), Aroma (Ar) and Basic Taste (BT). NS: Not significant at 5%, *: significant at 5%, **: significant at 1%, ***: significant at 0.1%.

Figure 3. Sensory profile of a fermented green tea infusion produced according to the process in Example 5. Results are compared among the fermented green teas fermented with an S. cerevisiae yeast strain HF13011 , HF13004 or HF13005, respectively. Modalities assessed include Flavor (FL), Mouthfeel (MF) and Aftertaste (AT). NS: Not significant at 5%, *: significant at 5%, **: significant at 1%, ***: significant at 0.1%.

Detailed Description of the Invention

Aspects and embodiments of the present invention will now be discussed with reference to the accompanying figures. Further aspects and embodiments will be apparent to those skilled in the art. All documents mentioned in this text are incorporated herein by reference.

Plant materials

The plant material used in the infusion described herein comprises a tea leaf, i.e. a leaf from a plant of the Camellia genus, preferably Camellia sinensis. For the avoidance of doubt, reference herein to the singular “leaf’ and the plural “leaves” are used interchangeably.

The plant material may comprise a white tea leaf, a black tea leaf and/or a green tea leaf. The plant material may comprise English breakfast tea, earl grey tea, pu-erh tea, jasmine tea, lapsang tea, matcha tea, oolong tea, Ceylon tea, Darjeeling tea, Assam tea and/or other Japanese style teas, such as gyokuro or sencha. The plant material may be a blend of multiple tea leaves, and therefore may comprise a combination of more than one of the tea leaves described above. For example, the plant material may comprise a combination of pu-erh and oolong tea leaves.

The plant material may comprise caffeine, for example the plant material may comprise about 0.001- 12.0%wt caffeine, preferably 0.1-10.0%wt caffeine, more preferably about 0.5-7%wt caffeine, even more preferably about 1.0-5%wt. Preferably, the plant material comprises at least 0.01%wt, 0.02%wt, 0.03%wt, 0.05%wt or at least 0.08%wt caffeine, more preferably at least 0.1 %wt, 0.2%wt, 0.3%wt, 0.4%wt or at least 0.5%wt caffeine. Preferably, the plant material comprises at least 0.7%wt, 1 %wt, 1.2%wt, 1.5%wt or at least 2%wt caffeine, more preferably at least 2.2%wt, 2.5%wt, 3%wt, 3.5%wt or at least 4%wt caffeine.

In some cases, the plant material may be decaffeinated. For example, any of the aforementioned plants may have their caffeine removed. Alternatively, such plants may be bred, for example by selecting breeding and/or by mutation, to reduce or remove the caffeine content to undetectable levels. In addition to the tea leaf, the plant material described herein may comprise material from other plants. For example, the plant material may further comprise material from one or more those selected from a group consisting of: Ilex genus (e.g. Ilex paraguariensis and Ilex guayusa); and Paullinia genus (e.g. Paullinia cupana). The plant material may comprise a leaf from Ilex paraguariensis and/or Ilex guayusa. The plant material may comprise a seed (e.g. a seed powder) from Paullinia cupana.

In particular embodiments, the plant material comprises a combination of a green tea leaf, an Ilex guayusa leaf, and a Paullinia cupana seed (e.g. a seed powder).

Infusions

Prior to fermentation, the plant material is prepared as an infusion. The term ‘infusion’ is commonly used, e.g. in the beverage industry, to refer to a drink made by soaking tea leaves, seeds, seed powder etc. in liquid, preferably water. As used herein, the term ‘plant material infusion’ refers to a liquid composition obtained by contacting a plant material defined herein (e.g. tea leaf) with water, preferably at an elevated temperature. When in the context of a tea leaf, an infusion is commonly referred to as “tea”.

The production of an infusion can be distinguished from methods in the prior art to produce ‘selective extracts’ of a specific plant material, wherein the plant material is e.g. boiled vigorously in water and/or other solvent(s), sometimes repeatedly i.e. over multiple extraction steps, and is often then further concentrated e.g. in vacuo, to maximise the yield of specific organic compounds removed from the plant. For example, catechin extracts can be prepared by selective extraction of this metabolite from tea leaves using methods known in the art. The infusions used in the present invention are distinct from these highly concentrated ‘selective extracts’ of plant material. In the latter, the ratios of chemicals in the extracted material have been highly modified in comparison to those found in nature

Preparation of the infusions described herein may comprise contacting (i.e. mixing, combining) the plant material with water at an elevated temperature (i.e. above about 40 °C).

Preparation of the infusions described herein may involve contacting the plant material with water at a temperature of greater than about 50 °C, more preferably greater than about 60 °C, even more preferably greater than about 70 °C.

Preferably, the infusion is produced at a temperature below boiling point (i.e. below 100 °C) such that organic compounds from the plant material (e.g. the flavour and aroma compounds) are gently dissolved into the water. Preparation of the infusions described herein may involve contacting the plant material with water at a temperature below about 95°C, or below about 90 °C. In some embodiments, the infusion may be prepared as a “cold infusion” at a temperature that is less than about 75 °C.

Preparation of the infusions described herein may involve contacting the plant material with water at a temperature between about 60 °C and about 100 °C, preferably between about 60 °C and about 95 °C, more preferably between about 60 °C and about 90 °C. Preparation of the infusions described herein may involve contacting the plant material with water at a temperature between about 70 °C and about 100 °C, preferably between about 70 °C and about 95 °C, more preferably between about 70 °C and about 90 °C, even more preferably between about 75 °C and about 85 °C. In particularly preferred cases, the temperature is about 80 °C.

The duration of the infusion step - i.e. the length of time during which the plant material is in contact with the (hot) water: the ‘steep’ time - is less than about 120 minutes, preferably less than about 90 minutes, more preferably less than about 75 minutes, even more less than about 60 minutes, yet more preferably less than about 45 minutes.

In some cases, the duration of the infusion step is longer than about 10 minutes, preferably longer than about 15 minutes.

In some cases, the duration of the infusion step is from about 10 to about 75 minutes, preferably from about 15 to about 60 minutes, more preferably from about 15 to about 45 minutes. In some cases, the duration of the infusion step is about 30 minutes.

In some embodiments, the infusion is produced by contacting plant material with water at a concentration (w/v) greater than about 0.5 g/L. In some embodiments, the infusion is produced by contacting plant material with water at a concentration (w/v) greater than about 1 g/L. In some embodiments, the infusion is produced by contacting plant material with water at a concentration (w/v) lower than about 50 g/L. In some embodiments, the infusion is produced by contacting plant material with water at a concentration (w/v) lower than about 40 g/L. In some embodiments, the infusion is produced by contacting plant material with water at a concentration (w/v) lower than about 30 g/L. In some embodiments, the infusion is produced by contacting plant material with water at a concentration (w/v) lower than about 20 g/L. In some embodiments, the infusion is produced by contacting plant material with water at a concentration (w/v) lower than about 10 g/L. In particular embodiments, where the plant material comprises a leaf from a plant of the Camellia genus (e.g. a green tea leaf), the infusion may be produced by the contacting tea leaf with water at a concentration (w/v) between about 0.5 and 10 g/L, e.g. between about 1 and 10 g/L, between about 2 and 10 g/L, between about 1 and 8 g/L, or between about 2 and 8 g/L. In some exemplary embodiments, the infusion is produced by contacting a leaf from a plant of the Camellia genus (e.g. a green tea leaf) with water at a concentration (w/v) of about 3 g/L.

Additional water may be added to the infusion of plant material prior to the fermentation step (for example, along with the microorganism or in a separate step, e.g. to top-up evaporated water after infusion or to dilute the infusion to a desired concentration). The concentrations above refer to the amount of plant material present during the ‘steep’ (infusion process).

In some embodiments, at least one carbohydrate is preferably added to the infusion of the plant material, to be used by the microorganism(s) as a carbon source during the fermentation reaction. The carbohydrate used is not particularly limited, so long as it is capable of being used by the microorganism(s) as a carbon source during fermentation. The carbohydrate preferably comprises one or more sugars, which include monosaccharides such as glucose, fructose and galactose, and disaccharides such as sucrose, lactose and maltose. Other carbohydrates include, but are not limited to, starch, cellulose, hemicelluloses, pectin, inulin, pullulan and saccharose. In some embodiments, the carbohydrate is a sugar, e.g. sucrose.

The carbohydrate may conveniently be added to the water along with the plant material, before or during the infusion process. Some or all of the carbohydrate may also be added to the microorganism, prior to its addition to the infusion. For example, in particular when the microorganism is a yeast, some sugar may be used to ‘activate’ the yeast, prior to its addition to the infusion.

The total amount of carbohydrate added to the infusion (i.e. the amount of carbohydrate present at the start of the fermentation step) is preferably more than about 5 g/L. In some embodiments, the total amount of carbohydrate added to the infusion is more than about 10 g/L. In some embodiments, the total amount of carbohydrate added to the infusion is more than about 15 g/L. In some embodiments, the total amount of carbohydrate added to the infusion is more than about 20 g/L. In some embodiments, the total amount of carbohydrate added to the infusion is more than about 25 g/L. In some embodiments, the total amount of carbohydrate added to the infusion is equal to or more than about 30 g/L.

The total amount of carbohydrate added to the infusion (i.e. the amount of carbohydrate present at the start of the fermentation step) is preferably less than about 60 g/L. In some embodiments, the total amount of carbohydrate added to the infusion is less than about 50 g/L. In some embodiments, the total amount of carbohydrate added to the infusion is less than about 40 g/L. In some embodiments, the total amount of carbohydrate added to the infusion is less than about 35 g/L.

In some embodiments, the total amount of carbohydrate added to the infusion is from 5 g/L to 50 g/L. In some embodiments, the total amount of carbohydrate added to the infusion is from 10 g/L to 40 g/L. In some embodiments, the total amount of carbohydrate added to the infusion is from 20 g/L to 50 g/L. In some embodiments, the total amount of carbohydrate added to the infusion is from 20 g/L to 40 g/L. In some embodiments, the total amount of carbohydrate added to the infusion is from 30 g/L to 50 g/L. In some embodiments, the total amount of carbohydrate added to the infusion is about 30 g/L.

In preferred embodiments, the carbohydrate feedstock used is a sugar, e.g. sucrose. One common way of measuring the approximate amount of sugars in beverages is to determine the Brix value. A Brix value, expressed as degrees Brix (°Bx), is a measure of the amount of dissolved substances present in a liquid. Soluble solids in the infusions and beverages described herein will comprise mainly sugars. One degree Brix is defined as one gram of sucrose in 100 grams of aqueous solution.

Brix can be measured using well known methods, for example using a refractometer or a hydrometer. A refractometer determines degrees Brix by measuring the refraction of light passing through a liquid sample. Liquids containing dissolved solids such as sugars are denser than water and cause greater refraction as light passes through. The instrument compares this to the refraction of light through water and provides the Brix value.

The total amount of sugar added to the infusion (i.e. the amount of sugar present at the start of the fermentation step) is preferably more than about 5 g/L (0.5 ⁰ Brix). In some embodiments, the total amount of sugar added to the infusion is more than about 10 g/L (1 ° Brix). In some embodiments, the total amount of sugar added to the infusion is more than about 15 g/L (1.5 ° Brix). In some embodiments, the total amount of sugar added to the infusion is more than about 20 g/L (2 ° Brix). In some embodiments, the total amount of sugar added to the infusion is more than about 25 g/L (2.5 ° Brix). In some embodiments, the total amount of sugar added to the infusion is equal to or more than about 30 g/L (3 ° Brix).

The total amount of sugar added to the infusion (i.e. the amount of sugar present at the start of the fermentation step) is preferably less than about 60 g/L (6 ° Brix). In some embodiments, the total amount of sugar added to the infusion is less than about 50 g/L (5 ° Brix). In some embodiments, the total amount of sugar added to the infusion is less than about 40 g/L (4° Brix). In some embodiments, the total amount of sugar added to the infusion is less than about 35 g/L (3.5 ° Brix).

In some embodiments, the total amount of sugar added to the infusion is from 5 g/L (0.5 ° Brix) to 50 g/L (5 ° Brix). In some embodiments, the total amount of sugar added to the infusion is from 10 g/L (1 ° Brix) to 40 g/L (4 ° Brix ). In some embodiments, the total amount of sugar added to the infusion is from 20 g/L (2 ° Brix) to 50 g/L (5 ° Brix). In some embodiments, the total amount of sugar added to the infusion is from 20 g/L (2 ° Brix) to 40 g/L (4 ° Brix). In some embodiments, the total amount of sugar added to the infusion is from 30 g/L (3 ° Brix) to 50 g/L (5 ° Brix). In some embodiments, the total amount of sugar added to the infusion is about 30 g/L (3 ° Brix).

Additional carbohydrates (e.g. sugars) may be added to the fermented infusion after the fermentation process has finished, e.g. to increase the sweetness of a beverage comprising the fermented infusion to the desired level. Further information regarding the production of beverages comprising the fermented infusion is provided below. The concentrations above refer to the amount of carbohydrate present at the start of the fermentation process and therefore available as a carbohydrate feedstock that can be used by the fermentation microorganism.

In some embodiments, the infusion is filtered to remove the plant material (e.g. the tea leaf), before the fermentation microorganism is added. In alternative embodiments, the plant material may be removed by other known methods such as e.g. centrifugation or decantation. References herein to filtration are intended to encompass also these methods, where appropriate. In other embodiments, the fermentation microorganism is added directly to the infusion without filtering: in some embodiments the plant material may then be removed at a later stage, along with other unwanted solids (e.g. biomass from the fermentation microorganism).

Provision of the infusions described herein may comprise preparation of the infusions as described above. Alternatively, provision of the infusions described herein may comprise purchasing the infusions as described above.

Fermentation

To produce a fermented infusion of the plant material, the infusion is subject to a fermentation step. Fermentation can be defined as a metabolic process by which a microorganism (e.g. a yeast, a fungus or bacteria) converts carbohydrate (i.e. starch or a sugar) into alcohol or acids and/or carbon dioxide. Fermentative modification of other organic compounds present in the substrate (fermentation medium) occurs concurrently, resulting in further changes to the chemical composition of the substrate. The term 'fermented', as used herein, may be construed accordingly and refers to a product which has been subjected to a fermentation process, by inoculation with a suitable microorganism, preferably in the presence of a suitable carbohydrate feedstock.

Preferably, the microorganism is added directly to the liquid product from the infusion step (after filtration, if applicable). Preferably, the infusion of a plant material prepared as set out above is used directly in the fermentation step which follows, except that in some embodiments the infusion may be filtered to remove plant material, and/or in some embodiments it may be diluted by the addition of further liquid (water). In particular, in preferred embodiments, the infusion is not subjected to any chemical modification, solvent extraction, or concentration steps prior to fermentation.

In some embodiments, additional water may be added, either separately or along with the microorganism (e.g. as part of a preparation of the microorganism). In some embodiments, the microorganism is added in a liquid preparation comprising additional water. In some embodiments, the microorganism is added in a liquid preparation comprising water and some or all of the sugar required for the fermentation.

In some embodiments, the microorganism used to ferment the infusion is a yeast.

For example, preferably the yeast may be selected from a group consisting of: Saccharomyces spp.; Pichia spp:, Zygosaccharomyces spp:, Kluyveromyces spp:, Kloeckera spp:, Brettanomyces spp.

Metschnikowia spp. Aureobasidium spp.; Issatchenkia spp.; Torulaspora spp. ; Lachancea spp. ; and Hanseniaspora spp..

Preferably, the yeast is selected from a group consisting of S. cerevisiae; S. uvarum; S. bayanus; S. exiguus; S. carlsbergensis; T. delbrueckii (formerly known as Saccharomyces delbrueckii, Saccharomyces rosei or Saccharomyces roseus); Lachancea thermotolerans; P. anomala; P. kluyveri; P. caribbica; P. guilliermondii; Z. bailii; K. marxianus; K. lactis; M. pulcherri. ma; A. pullulans, I. orientalis; K. apiculata; K. javanica; H. uvarum; H. osmophilia.

More preferably, the yeast is a member of the Saccharomyces sensu stricto group, or a derivative thereof. Most preferably, the yeast is selected from a group consisting of: S. cerevisiae; S. uvarum; S. bayanus; S. exiguus; S. carlsbergensis. It is especially preferred that the yeast is S. cerevisiae. The inventors identified that suitable microorganisms (e.g. yeasts) for producing fermented infusions with the low levels of acetic acid and alcohol described herein could be identified though a screening method. Details of the screening method are provided in Example 1.

Thus in some embodiments, the yeast (e.g. a S. cerevisiae yeast) may be capable of producing a test fermented green tea infusion with a pH of about 4 or less, less than about 7 g/L ethanol, less than about 0.5 g/L acetic acid, and less than about 5 g/L total sugar in a fermentation screening assay, the fermentation screening assay comprising preparing a test green tea infusion by contacting 10 g of green tea leaf with water at 80 °C for 45 minutes; adding 20 g/L sucrose or glucose (e.g. glucose) to 5 ml of the test green tea infusion; adding between 0.2 and 0.4 g/L (e.g. 0.2 g/L) of the yeast to the test green tea infusion; allowing the test green tea infusion to ferment at between 18 and 28 °C (e.g. 28 °C) for 6 days to produce the test fermented green tea infusion. pH, ethanol levels, acetic acid levels and sugar levels can be measured using methods known in the art. For example, pH can be measured using a suitable pH meter; ethanol levels can be measured using a K- ETOH Ethanol assay kit (Megazyme, Ireland) according to manufacturer’s instructions; acetic acid levels can be measured using a K-ACETRM acetic acid assay kit (Megazyme, Ireland) according to manufacturer’s instructions; and sugars can be measured using K-FRUGL D-Fructose/D-Glucose Assay Kit (Megazyme, Ireland) according to manufacturer’s instructions.

In preferred embodiments, the yeast is capable of producing a test fermented green tea infusion with a pH of about 4 or less, less than about 5 g/L ethanol, less than about 0.5 g/L acetic acid, and less than about 5 g/L total sugar in the fermentation screening assay.

In some embodiments, the fermentation screening assay may comprise rehydrating the dry yeast prior to adding it to the test green tea infusion. Rehydration may comprise resuspending the 10 g/L yeast and 10 g/L rehydration nutrient (e.g. commercial rehydration nutrients such as Natustart K or Go-Ferm Protect) at 23 °C or 37 °C (e.g. 37 °C) for 10-30 minutes.

In some embodiments, the fermentation microorganism added to the infusion of plant material does not comprise acetic acid bacteria. Acetic acid bacteria are bacteria within the family Acetobacteraceae and comprise those bacteria of the genera Acetobacter, Acidomonas, Ameyamaea, Asaia, Gluconacetobacter, Gluconobacter, Granulibacter, Kozakia, Neoasaia, Neokomagataea, Saccharibacter, Swaminathania and Tanticharoenia.

The fermentation step is carried out under conditions suitable for the microorganism(s) used, as is understood in the art.

In some embodiments, the fermentation step may be batch, fed-batch or continuous. In some embodiments, the fermentation may be performed under substantially anaerobic conditions. In some embodiments, the fermentation may be performed under substantially aerobic conditions.

In some embodiments, before addition to the infusion the microorganism may be activated. In some embodiments, in particular when the microorganism is a yeast, activation comprises mixing the microorganism with water and, preferably, adding a suitable amount of a carbohydrate (e.g. a sugar), in order to start the metabolic process of fermentation in the microorganism.

The resulting preparation of the microorganism (i.e. microorganism plus water, plus carbohydrate if applicable) is added directly to the infusion, to start the fermentation step of the process described herein. Any water and carbohydrate included in this preparation contribute to the overall water and carbohydrate content of the infusion, as noted elsewhere, and hence form part of the fermentation medium.

In some embodiments, for activation purposes, carbohydrate is added to the microorganism in a ratio from about 10:1 to about 50:1 (carbohydrate/yeast w/w). In some embodiments, carbohydrate is added to the microorganism in a ratio from about 15:1 to about 35:1 w/w. In some embodiments, carbohydrate is added to the microorganism in a ratio from about 20:1 to about 30:1 w/w. In some embodiments, carbohydrate is added in a ratio of about 25:1 w/w.

In some embodiments, the fermentation microorganism is present in an amount of at least about 0.1 g/L in the infusion (fermentation medium). In some embodiments, the fermentation microorganism is present in an amount of at least about 0.2 g/L. In some embodiments, the fermentation microorganism is present in an amount of at least about 0.3 g/L. In some embodiments, the fermentation microorganism is present in an amount of at least about 0.4 g/L. In some embodiments, the fermentation microorganism is present in an amount of about 0.4 g/L in the fermentation medium. In some embodiments, the fermentation microorganism is present in an amount of no more than about 0.6 g/L in the fermentation medium. In some embodiments, the fermentation microorganism is present in an amount of no more than about 0.8 g/L. In some embodiments, the fermentation microorganism is present in an amount of no more than about 1 g/L. In some embodiments, the fermentation microorganism is present in an amount of no more than about 2 g/L.

In some embodiments, the fermentation step is performed at a temperature of between 15 °C and 40 °C.

In some embodiments, the fermentation step is performed at a temperature of between 20 °C and 35 °C.

In some embodiments, the fermentation step is performed at a temperature of between 25 °C and 30 °C.

In some embodiments, the fermentation step is performed at a temperature of between 26 °C and 28 °C.

In some embodiments, the fermentation step is performed at a temperature below 35 °C. In some embodiments, the fermentation step is performed at a temperature below 32 °C. In some embodiments, the fermentation step is performed at a temperature below 30 °C. In some embodiments, the fermentation step is performed at a temperature above 15 °C. In some embodiments, the fermentation step is performed at a temperature above 20 °C. In some embodiments, the fermentation step is performed at a temperature above 25 °C.

In some embodiments, the duration of the fermentation is at least 2 hours. In some embodiments, the duration of the fermentation is at least 4 hours. In some embodiments, the duration of the fermentation is at least 24 hours. In some embodiments, the duration of the fermentation is at least 48 hours. In some embodiments, the duration of the fermentation is at least 72 hours (3 days). In some embodiments, the duration of the fermentation step may be less than 14 days. In some embodiments, the duration of the fermentation step may be less than 10 days. In some embodiments, the duration of the fermentation step may be less than 7 days. In some embodiments, the duration of the fermentation step may be less than 5 days. In some embodiments, the duration of the fermentation step may be less than 4 days.

In some embodiments, the duration of the fermentation step is about 1 day. In some embodiments, the duration of the fermentation step is about 2 days. In some embodiments, the duration of the fermentation step is about 3 days.

In some embodiments, the duration of the fermentation step is about 4 to 24 hours. In some embodiments, the duration of the fermentation step is about 8 to 22 hours. In some embodiments, the duration of the fermentation step is about 10 to 20 hours.

In some embodiments of the invention, the duration of fermentation is determined by the consumption of the carbohydrate feedstock (e.g. the sucrose). This can be monitored by methods which are known in the art (e.g. by measuring Brix). In some embodiments, fermentation is continued until the carbohydrate content is reduced by at least 10%, or at least 20%, or least 30% of the original carbohydrate feedstock. For example, where about 30 g/L carbohydrate was added to the fermentation reaction, fermentation may continue until about 20 g/L carbohydrate content remains in the fermented infusion. In some embodiments, at least 5 g/L carbohydrate, or at least 10 g/L carbohydrate is consumed by the microorganism during the fermentation reaction.

In some embodiments, fermentation is continued until the carbohydrate content is reduced by at least 50%, or at least 60%, or at least 70% or at least 80% of the original carbohydrate feedstock. For example, where about 1 g/L carbohydrate was added to the fermentation reaction, fermentation may continue until about 0.3 g/L carbohydrate content remains in the fermented infusion.

In some embodiments, the fermented infusion (i.e. at the end of fermentation) has a carbohydrate content of about 55 g/L or less, and/or a Brix value of about 5.5 ° Brix or less. In some embodiments, the fermented infusion has a carbohydrate content of about 50 g/L or less, and/or a Brix value of about 5 ° Brix or less. In some embodiments, the fermented infusion has a carbohydrate content of about 45 g/L or less, and/or a Brix value of about 4.5 ° Brix or less. In some embodiments, the fermented infusion has a carbohydrate content of about 40 g/L or less, and/or a Brix value of about 4 ° Brix or less. In some embodiments, the fermented infusion has a carbohydrate content of about 35 g/L or less, and/or a Brix value of about 3.5 ° Brix or less. In some embodiments, the fermented infusion has a carbohydrate content of about 3 g/L or less, and/or a Brix value of about 3 ° Brix or less.

In some embodiments, the fermented infusion has a carbohydrate content of between about 10 g/L and 60 g/L and/or a Brix value of between about 1 ° and 6 ° Brix. In some embodiments, the fermented infusion has a carbohydrate content of between about 15 g/L and 60 g/L and/or a Brix value of between about 1.5 ° and 6 ° Brix. In some embodiments, the fermented infusion has a carbohydrate content of between about 20 g/L and 60 g/L and/or a Brix value of between about 2 ° and 6 ° Brix.

In some embodiments, the fermented infusion has a carbohydrate content of between about 10 g/L and 55 g/L and/or a Brix value of between about 1 ⁰ and 5.5 ° Brix. In some embodiments, the fermented infusion has a carbohydrate content of between about 15 g/L and 55 g/L and/or a Brix value of between about 1.5 ⁰ and 5.5 ° Brix. In some embodiments, the fermented infusion has a carbohydrate content of between about 20 g/L and 55 g/L and/or a Brix value of between about 2 ° and 5.5 ° Brix.

In some embodiments, the fermented infusion has a carbohydrate content of between about 3 g/L and 20 g/L and/or a Brix value of between about 0.3 ° and 2.0 ° Brix. In some embodiments, the fermented infusion has a carbohydrate content of between about 3 g/L and 10 g/L and/or a Brix value of between about 0.3 ° and 1.0 ° Brix. In some embodiments, the fermented infusion has a carbohydrate content of between about 4 g/L and 9 g/L and/or a Brix value of between about 0.4 ° and 0.9 ° Brix.

In some embodiments, the fermented infusion has a carbohydrate content of between about 10 g/L and 50 g/L and/or a Brix value of between about 1 ° and 5 ° Brix. In some embodiments, the fermented infusion has a carbohydrate content of between about 15 g/L and 50 g/L and/or a Brix value of between about 1.5 ⁰ and 5 ° Brix. In some embodiments, the fermented infusion has a carbohydrate content of between about 20 g/L and 50 g/L and/or a Brix value of between about 2 ⁰ and 5 ° Brix.

In some embodiments, the fermented infusion has a carbohydrate content of between about 10 g/L and 45 g/L and/or a Brix value of between about 1 ° and 4.5 ° Brix. In some embodiments, the fermented infusion has a carbohydrate content of between about 15 g/L and 45 g/L and/or a Brix value of between about 1.5 ° and 4.5 ° Brix. In some embodiments, the fermented infusion has a carbohydrate content of between about 20 g/L and 45 g/L and/or a Brix value of between about 2 ° and 4.5 ° Brix.

In some embodiments, the fermented infusion has a carbohydrate content of between about 10 g/L and 40 g/L and/or a Brix value of between about 1 ° and 4 ° Brix. In some embodiments, the fermented infusion has a carbohydrate content of between about 15 g/L and 40 g/L and/or a Brix value of between about 1.5 ° and 4 ⁰ Brix. In some embodiments, the fermented infusion has a carbohydrate content of between about 20 g/L and 40 g/L and/or a Brix value of between about 2 ° and 4 ° Brix.

In some embodiments, the fermented infusion has a carbohydrate content of between about 10 g/L and 35 g/L and/or a Brix value of between about 1 ° and 3.5 ° Brix. In some embodiments, the fermented infusion has a carbohydrate content of between about 15 g/L and 35 g/L and/or a Brix value of between about 1.5 ° and 3.5 ° Brix. In some embodiments, the fermented infusion has a carbohydrate content of between about 20 g/L and 35 g/L and/or a Brix value of between about 2 ° and 3.5 ° Brix.

In some embodiments, the fermented infusion has a carbohydrate content of between about 10 g/L and 30 g/L and/or a Brix value of between about 1 ° and 3.0 ° Brix. In some embodiments, the fermented infusion has a carbohydrate content of between about 15 g/L and 30 g/L and/or a Brix value of between about 1.5 ° and 3 ° Brix. In some embodiments, the fermented infusion has a carbohydrate content of between about 20 g/L and 30 g/L and/or a Brix value of between about 2 ° and 3 ° Brix. In some embodiments of the invention, the duration of fermentation is determined by the pH of the fermented infusion. In some embodiments, fermentation is continued until the pH of the fermented infusion is less than about 4.

In some embodiments, the pH of the infusion at the start of the fermentation may be less than about 7. In some embodiments, the pH of the infusion at the start of the fermentation may be less than about 6.5. In some embodiments, the pH of the infusion at the start of the fermentation may be less than about 6. In some embodiments, the pH of the infusion at the start of the fermentation may be less than about 5.5.

In some embodiments, the pH of the infusion at the start of the fermentation may be between about 5 and

7. In some embodiments, the pH of the infusion at the start of fermentation may be between about 5 and 6.

In some embodiments, the pH of the fermented infusion (i.e. the infusion at the end of the fermentation step) may be less than about 6. In some embodiments, the pH of the fermented infusion may be less than about 5.5. In some embodiments, the pH of the fermented infusion may be less than about 5. In some embodiments, the pH of the fermented infusion may be less than about 4.5.

In some embodiments, the pH of the fermented infusion may be between about 3 and 6. In some embodiments, the pH of the fermented infusion may be between about 3.5 and 6. In some embodiments, the pH of the fermented infusion may be between about 3 and 5.5. In some embodiments, the pH of the fermented infusion may be between about 3 and 5. In some embodiments, the pH of the fermented infusion may be between about 3 and 4.5. In some embodiments, the pH of the fermented infusion may be between about 4.

Alcohol content of aqueous solutions is typically expressed as percentage ethanol by volume (% v/v). Methods of measuring alcohol content are known in the art and include spectroscopic measurements (e.g. as described in the examples).

In some embodiments, the alcohol content in the fermented infusion (i.e. the infusion at the end of the fermentation step) is less than about 1.2%. In some embodiments, the alcohol content in the fermented infusion is less than about 1%. In some embodiments, the alcohol content in the fermented infusion is less than about 0.75%. In some embodiments, the alcohol content in the fermented infusion is less than about 0.5%. The acetic acid content of aqueous solutions can be expressed as a percentage mass of acetic acid in the total mass of solution (% w/w). Methods of measuring acetic acid content are known in the art and include spectroscopy and high performance liquid chromatography - ion chromatography (HPLC-IC) as described in the examples. HPLC-IC is considered a more accurate method of measuring acetic acid than spectroscopy and therefore in preferred embodiments the acetic acid content is measured by ion chromatography.

In some embodiments, the acetic acid content in the fermented infusion (i.e. the infusion at the end of the fermentation step) is less than about 1% (w/w). In some embodiments, the acetic acid content in the fermented infusion is less than about 0.9% (w/w). In some embodiments, the acetic acid content in the fermented infusion is less than about 0.8% (w/w). In some embodiments, the acetic acid content in the fermented infusion is less than about 0.7% (w/w). In some embodiments, the acetic acid content in the fermented infusion is less than about 0.6% (w/w). In some embodiments, the acetic acid content in the fermented infusion is less than about 0.5% (w/w). In some embodiments, the acetic acid content in the fermented infusion is less than about 0.4% (w/w). In some embodiments, the acetic acid content in the fermented infusion is less than about 0.3% (w/w). In some embodiments, the acetic acid content in the fermented infusion is less than about 0.2% (w/w).

In some embodiments, the ethyl hexanoate content in the fermented infusion (i.e. the infusion at the end of the fermentation step) is 15 to 50 times higher than the b-damascenone content in the fermented infusion, e.g. when measured using a headspace sampler coupled to mass spectrometry (headspace GC/MS). In some embodiments, the ethyl hexanoate content in the fermented infusion is 20 to 40 times higher than the b-damascenone content in the fermented infusion. In some embodiments, the 2- phenylethyl acetate content in the fermented infusion is 3 to 10 times higher than the b-damascenone content in the fermented infusion. In some embodiments, the 2-phenylethyl acetate content in the fermented infusion is 4 to 9 times higher than the b-damascenone content in the fermented infusion. In some embodiments, the ethyl hexanoate content in the fermented infusion is 2 to 5 times higher than the ethyl acetate content in the fermented infusion. In some embodiments, the ethyl hexanoate content in the fermented infusion is 2 to 10 times higher than the benzeneacetaldehyde content in the fermented infusion. In some embodiments, the ethyl hexanoate content in the fermented infusion is 20 to 70 times higher than the limonene content in the fermented infusion. In some embodiments, the 2-phenylethyl acetate content in the fermented infusion is 6 to 15 times higher than the limonene content in the fermented infusion. In some embodiments, the 2-phenylethyl acetate content in the fermented infusion is 1.5 to 5 times higher than the 1-octen-3-ol content in the fermented infusion.

Beverages

The fermented infusions may be formulated as a beverage. As used herein, a beverage is a liquid that is suitable for consumption. The fermented infusions themselves may be considered beverages, i.e. a beverage may consist essentially of a fermented infusion described herein. More typically, the fermented infusions will be combined with other ingredients and subjected to additional process steps such as dilution with another liquid (e.g. water) in order to formulate the beverage. That is, a beverage will normally comprise the fermented infusion (which may be referred to as a “fermented base”) and one or more additional ingredients.

In some embodiments, the beverage further comprises fermented infusions of plant material other than a tea leaf. For example, the beverage may comprise the fermented infusion of the tea leaf, a fermented infusion of a different plant material, and, optionally one or more additional ingredients as described below. In some embodiments, the beverage comprises two or three, preferably three, of the group consisting of i) a fermented infusion of a green tea leaf; ii) a fermented infusion of a Ilex guayusa leaf; and iii) a fermented infusion of a Paullinia cupana seed (e.g. seed powder).

Where the beverage comprises a fermented infusion, the beverage preferably also comprises the same low acetic acid (e.g. less than about 1% (w/w) and low alcohol (e.g. less than 1.2% (v/v)) properties as found in the fermented infusions. Thus, the beverages described here may have any of the acetic acid contents and an ethanol contents reported above in the context of the fermented infusions.

The beverage may comprise (in addition to the fermented infusion) one or more additional ingredient selected from a list consisting of: a flavouring agent (e.g. a natural flavouring agent), a stabiliser, a preservative, an extract (e.g. fruit, malt, roots, ginger and/or acai), a sugar (e.g. sucrose), a fruit juice, a sweetener (e.g. stevia), a colouring agent (e.g. a natural colouring agent), a vitamin (e.g. a B vitamin), a mineral (e.g. magnesium), and an amino acid (e.g. L-theanine).

The flavouring agent may be a fruit flavouring and/or a herbal flavouring. Suitable fruit flavourings include berry flavourings (e.g. strawberry, raspberry, blackcurrant, etc.), tropical flavourings (e.g. mango, passionfruit, etc.) and citrus flavourings (e.g. orange, lemon, etc.).

The stabiliser may comprise a carrageenan or furcellaran. The carrageenan may be kappa carrageenan, iota carrageenan or lambda carrageenan. The carrageenan may be iota carrageenan. Alternatively, the stabiliser may comprise a suitable gum. The stabiliser may comprise locust bean gum, oat gum, guar gum, tragacanth, acacia gum, xanthan gum, karaya gum, tara gum, gellan gum or gum ghatti. The stabiliser may comprise iota carrageenan, xanthan gum or guar gum. The stabiliser may comprise xanthan gum. The amount of the stabiliser added may comprise between 0.1 and 10 grams (e.g. between 0.3 and 5 grams, between 0.4 and 5 grams, or between 0.5 and 5 grams) of stabiliser per litre of beverage.

The preservative may comprise an antioxidant. The preservative antioxidant may comprise an ascorbate, a tocopherol, a gallate, an erythorbate and/or a phosphate. The antioxidant may comprise an ascorbate. The ascorbate may comprise ascorbic acid, sodium ascorbate, calcium ascorbate, potassium ascorbate, fatty acid esters of ascorbic acid or ascorbyl stearate. In a preferred embodiment, the antioxidant comprises ascorbic acid (i.e. Vitamin C). The amount of the antioxidant added may comprise between 0.1 and 10 grams (e.g. between 0.25 and 5 grams, between 0.5 and 2 grams, or between 0.5 and 5 grams) of antioxidant per litre of beverage.

The preservative may comprise an antimicrobial agent. The antimicrobial agent may comprise an antifungal agent and/or an antibacterial agent. Preferably, the antimicrobial agent comprises an antifungal agent and an antibacterial agent. The antifungal agent may comprise a sorbate. The sorbate may comprise sorbic acid, sodium sorbate, potassium sorbate or calcium sorbate. The antimicrobial agent may comprise potassium sorbate. The amount of the antimicrobial agent added may comprise between 100 and 3000 milligrams (e.g. between 200 and 2500 milligrams, between 500 and 2250 milligrams, or between 750 and 2000 milligrams) of antimicrobial agent per litre of beverage.

In some embodiments, a sugar (e.g. sucrose) may be added to the beverage. The amount of sugar added will depend on the sugar content of the fermented infusion(s) present in the beverage and any other ingredients that contain sugar (e.g. fruit juice). In some embodiments, the beverage may comprise a carbohydrate (e.g. sugar) content of less than about 80 g/L (less than about 8 ° Brix), less than about 70 g/L (less than about 7 ° Brix), less than about 60 g/L (less than about 6 ° Brix), less than about 50 g/L (less than about 5 ° Brix). In some embodiments, the beverage may comprise a carbohydrate content that is greater than about 5 g/L (greater than about 0.5 ° Brix), greater than about 10 g/L (greater than about 1 ° Brix), greater than about 15 g/L (greater than about 1.5 ° Brix), or greater than about 20 g/L (greater than about 2 ° Brix).

In some embodiments, beverage may comprise a carbohydrate content that is between about 5 g/L and about 80 g/L (between about 0.5 ° and about 8 ° Brix). In some embodiments, beverage may comprise a carbohydrate content that is between about 10 g/L and about 80 g/L (between about 1 ° and about 8 ° Brix). In some embodiments, beverage may comprise a carbohydrate content that is between about 15 g/L and about 80 g/L (between about 1.5 ° and about 8 ° Brix). In some embodiments, beverage may comprise a carbohydrate content that is between about 20 g/L and about 80 g/L (between about 2 ° and about 8 ° Brix).

In some embodiments, beverage may comprise a carbohydrate content that is between about 5 g/L and about 70 g/L (between about 0.5 ° and about 7 ° Brix). In some embodiments, beverage may comprise a carbohydrate content that is between about 10 g/L and about 70 g/L (between about 1 ° and about 7 ° Brix). In some embodiments, beverage may comprise a carbohydrate content that is between about 15 g/L and about 70 g/L (between about 1.5 ° and about 7 ° Brix). In some embodiments, beverage may comprise a carbohydrate content that is between about 20 g/L and about 70 g/L (between about 2 ° and about 7 ⁰ Brix). In some embodiments, beverage may comprise a carbohydrate content that is between about 5 g/L and about 60 g/L (between about 0.5 ° and about 6 ⁰ Brix). In some embodiments, beverage may comprise a carbohydrate content that is between about 10 g/L and about 60 g/L (between about 1 ⁰ and about 6 ° Brix). In some embodiments, beverage may comprise a carbohydrate content that is between about 15 g/L and about 60 g/L (between about 1.5 ° and about 6 ° Brix). In some embodiments, beverage may comprise a carbohydrate content that is between about 20 g/L and about 60 g/L (between about 2 ° and about 6 ° Brix).

In some embodiments, beverage may comprise a carbohydrate content that is between about 5 g/L and about 50 g/L (between about 0.5 ° and about 5 ° Brix). In some embodiments, beverage may comprise a carbohydrate content that is between about 10 g/L and about 50 g/L (between about 1 ° and about 5 ° Brix). In some embodiments, beverage may comprise a carbohydrate content that is between about 15 g/L and about 50 g/L (between about 1.5 ° and about 5 ° Brix). In some embodiments, beverage may comprise a carbohydrate content that is between about 20 g/L and about 50 g/L (between about 2 ° and about 5 ° Brix).

In some embodiments, the beverage comprises a sweetener (i.e. a sugar substance that provides sweetness). Known sweeteners include stevia (e.g. stevia leaves, a stevia extract, or fermented stevia), acesulfame potassium, aspartame, cyclamate, mogrosides, saccharin, sucralose and/or sugar alcohol. In some embodiments, the sweetener comprises fermented stevia.

In some embodiments, the beverage comprises a sweetener in an amount that provides a sweetness in the beverage equivalent to any of the Brix values referred to above in the context of a carbohydrate (e.g. sugar) content in the beverage. For example, the beverage may comprise a sweetener in an amount that, together with any sugar present in the beverage, provides a sweetness in the beverage equivalent to between about 1 ° and 7 ° Brix, e.g. between about 2 ° and 6 ° Brix. This may be useful in reducing the amount sugar that is added to the beverage. Since excess sugar is known to impact on obesity, diabetes and cardiovascular health, keeping the levels of sugar low in the beverage may be desirable when seeking to produce a healthier beverage.

In some embodiments, the beverage may be a low sugar beverage, e.g. the beverage may comprise a sugar content that is less than about 5 g/L or less. In such cases, the low sugar beverage may comprise a sweetener in an amount that provides a sweetness in the beverage equivalent to greater than 1 °, 2 °, or 3 ° Brix; or between about 1 ° and 6 ° Brix.

In some embodiments, the beverage comprises juice (e.g. fruit juice). The juice may be juice from one or more fruits selected from: pineapple, kiwi, apple, pear, coconut, cucumber, strawberry, raspberry, cherry, blueberry, blackcurrant, blackberry, plum, sour plum, hawthorn berry gooseberry, acai berry, guava, prickly pear, agave, tomato, hibiscus, grape, goji, yuzu, ananas, mango, melon, and/or citrus fruit. Melon may comprise water melon, papaya, galia melon, cantaloupe melon and/or honeydew melon. Citrus fruit may comprise orange, lemon, lime and/or grapefruit. The beverage may be carbonated. The carbonation may be natural (as a result of the fermentation with the fermentation microorganism), or it may be created by contacting with carbon dioxide.

The fermented infusion may be diluted with liquid, e.g. with water, in the beverage. The degree of dilution required will depend on how concentrated or strong the fermented infusion is.

In some embodiments, beverage contains a fermented infusion in an amount that is at least 25%, at least 50%, at least 75%, at least 80%, or at least 85% of the total weight of the beverage. In particular embodiments, the beverage contains the fermented infusion in an amount that is at (east 90% of the total weight of the beverage. Liquid (e.g. water) and the additional ingredients described above typically make up the remainder of the beverage. In some embodiments, beverage contains a fermented infusion in an amount of 10 to 40%, 15 to 35% or 20 to 30% of the total weight of the beverage.

Where the beverage comprises a combination of fermented infusions made from different plant materials, the sum of the combination of fermented infusions may be present in an amount that is at least 50%, at least 75%, at least 80%, at least 85%, or at least 90% of the total weight of the beverage. In some embodiments, the sum of the combination of fermented infusions is present in an amount that is at least 10% , at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40% or at least 45%, of the total weight of the beverage.

Preferably, the pH of the (diluted) beverage is less than about 4. Advantageously, such pHs offer protection to microbial spoilage. It will be appreciated that the pH of the diluted beverage may be raised due to the presence of water following the dilution step. In some embodiments the process of preparing a beverage from the fermented infusion may comprise the addition of a pH modulator, such as lemon juice, to the fermented infusion and/or beverage (depending on which stage the modulator is added).

In some embodiments the process of preparing a beverage from the fermented infusion may comprise pasteurising the fermented infusion and/or the beverage. It will be appreciated that pasteurisation is a combination of heat and time, and 70°C for about 5 minutes are believed to be suitable conditions for the present invention.

The features disclosed in the foregoing description, or in the following claims, or in the accompanying drawings, expressed in their specific forms or in terms of a means for performing the disclosed function, or a method or process for obtaining the disclosed results, as appropriate, may, separately, or in any combination of such features, be utilised for realising the invention in diverse forms thereof.

While the invention has been described in conjunction with the exemplary embodiments described above, many equivalent modifications and variations will be apparent to those skilled in the art when given this disclosure. Accordingly, the exemplary embodiments of the invention set forth above are considered to be illustrative and not limiting. Various changes to the described embodiments may be made without departing from the spirit and scope of the invention.

For the avoidance of any doubt, any theoretical explanations provided herein are provided for the purposes of improving the understanding of a reader. The inventors do not wish to be bound by any of these theoretical explanations.

Any section headings used herein are for organizational purposes only and are not to be construed as limiting the subject matter described.

Throughout this specification, including the claims which follow, unless the context requires otherwise, the word “comprise” and “include”, and variations such as “comprises”, “comprising”, and “including” will be understood to imply the inclusion of a stated integer or step or group of integers or steps but not the exclusion of any other integer or step or group of integers or steps.

It must be noted that, as used in the specification and the appended claims, the singular forms “a,” “an,” and “the” include plural referents unless the context clearly dictates otherwise. Ranges may be expressed herein as from “about” one particular value, and/or to “about” another particular value. When such a range is expressed, another embodiment includes from the one particular value and/or to the other particular value. Similarly, when values are expressed as approximations, by the use of the antecedent “about,” it will be understood that the particular value forms another embodiment. The term “about” in relation to a numerical value is optional and means for example +/- 10%.

Examples

EXAMPLE 1 - Screening assay to identify suitable microorganisms for use in the fermentation process

The aim of this experiment was to develop a screening assay capable of identifying suitable microorganisms that were capable of producing a fermented infusion of a plant material having a low alcohol, low acetic acid and low sugar content.

The screening assay was performed on Saccharomyces cerevisiae wine yeast. Suitable wine yeasts that can be screened using this assay are commercially available. For example, wine yeast can be purchased from commercial entities such as WeissBioTech GmbH, Max Baldinger AG, Brouwland and local wine making suppliers. The yeasts identified below (HF13011 , HF13004, HF13005 and HF13019) were obtained from WeissBioTech GmbH.

Fermentation base- exemplary methodology

Green Tea production:

1 L of hot water (80 °C) is used to steep 10 g of green tea leaf for 45 minutes. After 45 minutes the tea steep is poured through a metal tea strainer and subsequently placed in a water bath at the desired fermentation temperature.

Yeast inoculum - exemplary methodology Rehydration Nutrients: Natustart K (Oenobiotech, France) or Go-Ferm Protect (Lallemand, France).

Rehydration Procedure:

Active dry yeast is re-hydrated by resuspending 10 g/L yeast and 10 g/L rehydration nutrient in 23 °C (for parallel fermentation tests at between 18 °C and 28 °C) or 37 °C (for tests at 28 °C) followed by incubation for 10-30 min.

Yeast fermentation setup - exemplary methodology

Fermentation Nutrients: Natustart Plus (Oenobiotech, France), Fermaid O (Lallemand, France) Headspace vials 10 ml ND18 (Fisher Scientific)

Screw Caps magnetic ND18 (Neolab)

Canula FINEJECT G26 0.45 x 12 mm (VWR)

Fermentation procedure:

Rehydrated yeast is added (final titer 0.2 - 0.4 g/L) to 5 ml green tea supplemented with granulated white table sugar (sucrose) or glucose (20 g/L) in 10 ml vials, which are subsequently closed with screw caps and inserted canula. The vials are incubated at the temperature of interest (18-28 °C) with (140 rpm) or without shaking for 6 days. At the end of fermentation, samples are pasteurized and analysed for residual sugar, ethanol and acetic acid properties.

Analytics - exemplary methodology

Acetic acid measurements were carried out using the K-ACETRM acetic acid assay kit (Megazyme, Ireland).

Ethanol measurements were carried out using the K-ETOH Ethanol assay kit (Megazyme, Ireland) Glucose and fructose measurements were carried out using the K-FRUGL D-Fructose/D-Glucose Assay Kit (Megazyme, Ireland). pH was measured using a WTW bench Routine meter pH 526.

The assay kits were used according to manufacturer’s instructions.

Results

Yeast were sought that could produce a fermented product in the assay having the following characteristics:

• less than 5 g/L total sugar;

• less than 7 g/L ethanol;

• less than 0.5 g/L acetic acid; and

• reach pH <4 without the addition of pH lowering agents.

It was also considered advantageous if the yeast was able to produce a fermented product in the assay having an ethanol content of less than 5 g/L. Accordingly, preferably the yeast is capable of producing a fermented product in the assay having the following characteristics: • less than 5 g/L total sugar;

• less than 5 g/L ethanol;

• less than 0.5 g/L acetic acid; and

• reach pH <4 without the addition of pH lowering agents.

Results for a selection of yeasts screened using the assay described in this example are provided below in Table 1. Fermentation in all cases was carried out for 6 days without shaking.

Table 1:

As can be seen from Table 1, this assay identified yeast strains HF13011 , HF13004 and HF13005 that produced fermented products with the desired < 5 g/L total sugar, < 7 g/L ethanol, < 0.5 g/L acetic acid and a pH of 4 or less. Yeast strain HF13019 produced a fermented product with an ethanol content of greater than 7 g/L and was not analysed further. Yeast strain HF13011 was of particular interest, as it produces a fermented product having less than 5 g/L ethanol.

EXAMPLE 2 - Comparison of fermentation products produced by yeast strains identified in Example 1

An experiment was carried out to characterise fermented green tea produced by each of the three yeast strains selected based on the results obtained in Example 1 (yeast strains HF13011, HF13004 and HF13005).

Materials & Methods

Green Tea production:

1200 mL of hot water (90 °C) was used to steep 12 g green tea leaf together with 1.428 g stevia dry leaf for 23 minutes. After 23 minutes, the tea steep was poured through the tea strainer and divided up into 3 containers, 300 mL each. 300 mL cold water was added to each tea preparation.

30 grams of sucrose (Tate and Lyle, UK) was dissolved in the tea at 30 g/L (approximately 3 brix). 0.4 g/L of the yeast strains HF13011 , HF13004 and HF13005 were added to each container, respectively. The fermentations were carried out at 30 °C and the Brix, pH, ethanol and acetic acid properties measured at various time points.

Analytics pH was measured using a Mettler Toledo Seven Easy pH Meter.

Acetic acid and alcohol measurement by spectroscopic measurement using Acetoscan (Cetotec, Germany).

Brix measurement by RFM340+ Digital Refractometer (B+S, A Nova Analytics company, UK) (range 0- 20° Brix, accuracy ±0.03).

Results

The Brix, pH, ethanol and acetic acid content measured after 2 and 3 days of fermentation using the three yeast strains are summarised in Table 2 below.

Table 2:

As demonstrated in the above table, strains HF13011 , HF13004 and HF13005 were able to produce fermented infusions with a Brix-content less than 3, an ethanol content less than 1.2%, an acetic acid content less than 1% after 2 and 3 days of fermentation. Increasing fermentation time from 2 days to 3 days increased the levels of ethanol and acetic acid produced and decreased the amount of sugars present in the fermented infusion (measured according to Brix), as expected.

Strain HF13005 produced a fermentation product with higher alcohol (but still less than 1.2%) than strains HF13011 and HF13004 after both 2 and 3 days of fermentation. The presence of stevia in the fermentations did not significantly affect the Brix, ethanol, acetic acid, or pH levels of the fermented infusions (data not shown). These results demonstrates that microbes identified according to the screening assay described in Example 1 can be used to produce fermented infusions having the desired low sugar, low alcohol and low acetic acid characteristics.

EXAMPLE 3 - Reproduction of the EP0791296B1 process

An experiment was carried out to produce a fermented sweet tea using the process disclosed in Example 2 of EP0791296B1 . The aim of this experiment was to identify the sugar, acetate and ethanol content in the fermented sweet tea produced using this prior art process. Additionally, the fermented sweet tea produced was tasted by a panel of testers to provide an indication of the sensory characteristics of this tea.

The green tea infusion was produced as described in Example 2 of EP0791296B1. Briefly, 1 % green tea was steeped in boiling water for 20 minutes, the green tea filtered and allowed to cool to 30 °C before 7% sucrose added to the aqueous solution. The pH of the infusion was adjusted to pH 5 using acetic acid.

The infusion was then inoculated with 0.05% S. cerevisiae yeast and 0.5% G. suboxydans bacteria. Two G. suboxydans strains used tested: a DSM50049 strain (as disclosed in EP0791296B1) and a DSM7145 strain. Both strains were obtained from DSMZ deposit. Two typical S. cerevisiae yeast wine yeast strains were used, the Vin-O-Ferm Thiols strain (“Yeast 1”) and the Vin-O-Ferm Vb Arom strain (“Yeast 2”).

Both yeast strains are commercially available, e.g. from OenoBioTech I WeissBioTech. Fermentations were carried out under aerobic conditions as set out in Table 3 below.

Table 3:

At the end of fermentation, the temperature of the fermented infusions was reduced to 5 °C, the solids removed by filtration and the solution pasteurized at 95 °C for 5 minutes.

The levels of glucose, fructose, acetate and ethanol were measured by high performance liquid chromatography (HPLC).

• Instrumentation:

- Agilent 1200 HPLC-DAD coupled to Rl detector

- BIORAD Aminex HPX-87H, 300x7,8 mm, 9 pm particles (anion exchange/ligand exchange chromatography) • Mobile Phase:

- Isocratic 30 mM H2SO4 + 1% AcN

- Flow: 0.6 ml/min

• Injection volume 20 pl (MTP format)

• Column temperature: 60 °C

• Total run time: 25 min per sample

The results obtained from the HPLC analysis are provided below in Table 4.

Table 4: nd - not detected

Neither acetate nor ethanol were detected by HPLC in any of the samples. This result is consistent with the very low levels of acetic acid and ethanol (0.032% and 0.05%, respectively) reported in EP0791296B1. The analysis focused on detecting minor metabolites of a fermentative origin, meaning that the accuracy of the measurements for the high content metabolites (such as the sugars) was reduced. However, in all cases very high levels of the sugars glucose and fructose (the monosaccharides components of the sucrose disaccharide) were reported. This demonstrates that most of the initial sugar is still retained in the final product.

The fermented green teas produced using this prior art method were also tested by a panel of tasters. The tasters provided the comments on these fermented teas that included the following: “sugary, thick”, “did not really taste like apple, more like grape, honey”, “mouthfeel sugary”, “really sweet”, “fermented notes are unpleasant: lactic taste, bready note”, "chemical taste in retronasal”, and “some fruit note but more grape”.

Volatile analysis was also carried out on the fermented green tea produced using this prior art method, as described in Example 4 below.

EXAMPLE 4 - Production of low alcohol, low acetic acid fermented teas

Materials and methods

Production: A green tea (GT) and a pu-erh and oolong blend (PO) were produced by steeping 3 g/L of tea leaf (see below Table 5) for 20 minutes starting at 80 °C.

Granulated white table sugar was added to all teas at 30 g/litre and an S. cerevisiae yeast strain HF13011 was added to all fermentations at 0.4 g/litre.

The dried yeast was rehydrated and activated in water and sugar for 2 hours at 30 °C before addition.

The teas were transferred to a 30 °C hot room and left to ferment for 2 days.

Table 5:

Analytics:

Daily measurements of pH, brix, density, alcohol and acetic acid were monitored. Density was measured using an Anton Paar DMA 4500M density meter. pH, Brix, ethanol and acetic acid was measured as described for Example 2.

Sensory analysis

Sensory tests are performed using a Quantitative Descriptive Analysis (QDA). A QDA evaluates if there are differences between samples and what the differences are (qualitative = description element, and by how much the samples differ= quantitative element). This methodology is run with sensory panellists who are a group of consumers who have been screened for their sensory acuity and trained on their senses to articulate what they perceive when consuming a beverage. They have also been trained on a range of sensory methodologies. The measure obtained from a sensory panel is objective. A minimum of 6-7 panellists participate in the studies and results are taken in duplicate to ensure reproducibility.

The samples are presented first all at a time to the sensory trained panel who, during a round table discussion, initially agree on the sensory attributes or descriptors which best describe the products, in order to develop a sensory vocabulary. A sensory scientist moderates the discussion and collects the attributes selected by the panel which are clear for them, easily defined and describe differences between the samples under study. Then sensory evaluation takes place in sensory booths where each panellist assesses the samples individually. The panellists rate one sample at a time in an undefined line scale from Low to High (0-100 of each attribute). The samples are presented in a randomised and balanced order to avoid first order and carry over effects. The data collection is done online via FIZZ (Sensory Software) and the data analysis carried out with FIZZ and XLSTAT. The outcome of the data analysis is a spider graph (see e.g. Figure 1). Statistical Significance testing in the form of ANOVA (Analysis of Variance) and an LSD test is performed. This determines on which attributes the samples are significantly different (if any). P-value is taken as < 0.05 (using 95% confidence).

Volatiles Analysis HPLC Method

Extraction of volatiles by Headspace Solid Phase Micro Extraction (SPME) for GC/MS Analysis

An appropriate amount of sample was transferred to a 20 ml headspace vial. All samples were analysed in triplicate. Vials were equilibrated at 75°C for 5 minutes with agitation. The headspace of the vial was then sampled for 5 minutes at 75°C with agitation using carboxen/ polydimethylsiloxane Zdivinylbenzene coated SPME fibre. The volatiles adsorbed onto the fibre were analysed by thermal desorption at 270°C in the injector port of the GC/MS.

GC/MS Analysis of volatiles

Analysis was carried out an Agilent 7890A gas chromatograph (GC) and Agilent 7200 accurate mass Q- TOF mass spectrometer (MS) via CTC Combi-Pal autosampler.

GC/MS conditions were as follows:

Column : 30mm x 0.25mm fused silica with ZB-semivolatiles stationary phase

Helium carrier gas flow rate: 1 mL mim ¹

Injector temperature: 270 C

Column temperature: 5 min at 40°C; then 4°C mim ¹ to 200°C; then 30°C min-1 to 350°C, hold for 3 minutes

MS analysis mode: Scam (33-350 m/z)

Peaks were tentatively identified by spectral matching with the NIST library of mass spectral data.

Results

Monitoring of fermentation reaction

The analytical results obtained from the daily measurements of pH, brix, density, alcohol and acetic acid are reported in Table 6 below.

Table 6:

Sensory profile

The sensory profile of the fermented green tea infusion produced using this method is illustrated in Figure 1. This figure illustrates differences in sensory characteristics perceived for the green tea infusion fermented using S. cerevisiae strain HF13011 (“fermented base”) and the unfermented green tea infusion (“unfermented base infusion”).

As demonstrated in Figure 1 , the green tea infusion fermented using S. cerevisiae strain HF13011 was perceived as having a more fermented aroma and a more fermented aftertaste compared to the unfermented green tea infusion. The green tea infusion fermented using strain HF13011 was also perceived as having a less tea aroma, a less tea flavour, a less bitter flavour, and a less tea aftertaste compared to the unfermented green tea infusion. The increase in fermented sensory characteristics and decrease in tea sensory characteristics demonstrates that the S. cerevisiae yeast modulates the sensory profile of the green tea infusion.

The green tea infusion fermented using S. cerevisiae strain HF13011 was additionally perceived as having an increased vinegar aroma, an increased vinegar flavour, and an increased vinegar aftertaste compared to the unfermented green tea infusion. Consistent with the low levels of acetic acid produced in the fermented green tea infusions, the increases in vinegar aroma, flavour and aftertaste were not as pronounced where compared to a green tea infusion fermented using kombucha. As described above, kombucha typically contains relatively high levels of acetic acid (>1 %), which accounts for the polarising and in some cases undesirable taste.

Organic acids analysis

High performance liquid chromatography-ion chromatography (HPLC-IC)

HPLC-IC was used to determine and quantify the organic acids in the fermented infusions of green tea produced using S. cerevisiae strain HF13011. This method produces a more accurate reading of organic acid content when compared to, e.g. the spectroscopic measurements reported above.

The sample is extracted using a basic water solution and the extract analysed by HPLC-IC. The quantification is performed using external standards. The parameters are provided as follows:

The organic acid results are set out in Table 7 below. Table 7

These results demonstrated that the levels of all organic acids tested in the fermented combination were below the lower limit of detection. For example, this demonstrates that the level of acetic acid present in the fermented combination was less than 0.2 g/ 100 g (0.2% (w/w)).

Volatile analysis

Volatiles analysis was performed using analytical HPLC as described above. Analysis was carried out on the green tea infusion fermented using S. cerevisiae strain HF13011 described above in Example 4 and on the fermented green tea produced using the prior art EP0791296B1 method described above in Example 3.

Analysis was carried out to identify those volatile compounds present and to identify those volatiles that were increased or decreased in the green tea infusions fermented using S. cerevisiae strain HF13011 compared to the green tea infusion fermented using the prior art method.

More than 190 volatiles were identified in the HF13011-fermented green tea, compared to the 90 volatiles identified in the green tea infusion fermented using the prior art method. It is believed that a greater number of volatiles present in the HF13011-fermented infusion is reflective of a more complex sensory profile (e.g. taste) when compared to the green tea infusion fermented using the prior art method. This is consistent with the understanding that only a limited amount of fermentation is taking place in the prior art fermentation process. A selection of volatiles that were increased or decreased in the green tea infusions fermented using S. cerevisiae strain HF13011 compared to the green tea infusion fermented using the prior art method is provided below. Compounds decreased in HF13011 -fermented green tea compared to green tea fermented using prior art process:

Compounds increased in HF13011 -fermented green tea compared to green tea fermented using prior art process:

EXAMPLE 5 - Production of low alcohol, low acetic acid fermented teas with various yeasts

Materials and methods Production:

A green tea (GT) was produced by steeping 3 g/L of tea leaf (see below Table 8) for 20 minutes starting at 75 °C.

Granulated white table sugar was added to all teas at 30 g/litre and an S. cerevisiae yeast strain HF13011 , HF13004 or HF13005 was added to all fermentations at 0.4 g/litre, respectively.

The dried yeast strain HF13011 , HF13004 and HF13005 were rehydrated and activated in water and sugar for 2 hours at 30 °C before addition.

The teas were transferred to a 30 °C hot room and left to ferment for 2 days.

Table 8:

Analytics:

Daily measurements of pH, brix, density, alcohol and acetic acid were monitored. Density was measured using an Anton Paar DMA 4500M density meter. pH, Brix, ethanol and acetic acid was measured as described for Example 2.

Sensory analysis

Profiling

5 sensory trained panellists evaluated the samples. The sensory profile started with an individual testing of all the samples. From this testing a vocabulary was generated and later was discussed by the panel. After the discussion, there was an agreement of the different attributes that define the sensory characteristics of the samples, focusing on the differences between them.

Selected attributes included modalities such as Appearance, Aroma, Flavour, Basic Taste, Mouthfeel and Aftertaste.

Sample assessment Samples were presented to the panel following a randomized and balanced design to avoid first order carryover effect. Individually the panelists evaluated the intensity of the selected attributes in a 100-point line scale (being 0 no intense to 100 very intense). This process was performed in duplicate to ensure the reproducibility of the panel and robust data.

Data analysis

For the results, statistical analysis was carried out on the data using ANOVA (Analysis of Variance) and Fisher’s LSD (Least Significant Difference) at a 95% confidence. This analysis distributed samples into different groups according to the significant differences among samples means. Different letters indicated the presence of a significant difference.

Results

Monitoring of fermentation reaction

The analytical results obtained from the daily measurements of pH, brix, density, alcohol and acetic acid are reported in Table 9 below.

Table 9:

Acetic acid and alcohol measurement was conducted by spectroscopic measurement using Acetoscan (Cetotec, Germany).

Sensory profile

The sensory profile of the fermented green tea infusions produced using this method is illustrated in Figures 2 and 3. These figures illustrate differences in sensory characteristics perceived for the green tea infusions fermented using S. cerevisiae strain HF13011 , HF13004 or HF13005.

As demonstrated in Figures 2 and 3, the green tea infusion fermented using S. cerevisiae strain HF13004 was perceived as having more cloudy, having more overall aroma, more yeasty aroma and flavor notes and less fermented flavour than the green tea infusion fermented using S. cerevisiae strain HF13011. The green tea infusion fermented using strain HF13005 was perceived as having a slightly darker gold colour, less dried fruits aroma, less yeasty aroma and flavour, more fermented notes, and less bitter in aftertaste compared to the green tea infusion fermented using S. cerevisiae strain HF13011.

Organic acids analysis

High performance liquid chromatography-ion chromatography (HPLC-IC)

HPLC-IC was used to determine and quantify the organic acids in the fermented infusions of green tea produced using S. cerevisiae strain HF13011, HF13004 or HF13005. This method produces a more accurate reading of organic acid content when compared to, e.g. the spectroscopic measurements reported above.

The sample is extracted using a basic water solution and the extract analysed by HPLC-IC. The quantification is performed using external standards. The parameters are provided as follows:

The organic acid results are set out in Table 10 below. Table 10

These results demonstrated that the levels of all organic acids tested in the fermented combination were below the lower limit of detection. For example, this demonstrates that the level of acetic acid present in the fermented combination was less than 0.2 g/ 100 g (0.2% (w/w)).

Exemplary embodiments

The present invention encompasses the following listing of exemplary embodiments: 1. A fermented infusion of a plant material comprising a tea leaf, wherein the fermented infusion has:

- an acetic acid content of about 1 % (w/w) or less; - an ethanol content of about 1.2% (v/v) or less; and

- a carbohydrate content of about 60 g/L or less, and/or a Brix value of about 6 ° Brix or less.

2. A fermented infusion according to embodiment 1, wherein the beverage has a carbohydrate content of between about 20 g/L and about 60 g/L, and/or a Brix value of between about 2 ° Brix and 6 ° Brix.

3. A fermented infusion according to embodiment 1 or embodiment 2, wherein the fermented infusion has a carbohydrate content of about 50 g/L or less, and/or a Brix content of about 5 ⁰ Brix or less.

4. A fermented infusion according to any one of the preceding embodiments, wherein the fermented infusion has an acetic acid content of about 0.5% (w/w) or less, or an acetic acid content of about 0.3% (w/w) or less; and/or wherein the fermented infusion has an ethanol content of about 1% (v/v) or less, or an ethanol content of about 0.5% (v/v) or less.

5. A fermented infusion according to any one of the preceding embodiments, wherein the plant material comprises a green tea leaf.

6. A fermented infusion according to any one of the preceding embodiments, wherein the fermented infusion is produced by

(a) preparing an infusion of the plant material; and

(b) adding a fermentation microorganism to the infusion, optionally wherein the fermentation microorganism is a yeast.

7. A fermented infusion according to embodiment 6, wherein the yeast comprises a Saccharomyces cerevisiae yeast.

8. A fermented infusion according to embodiment 6 or embodiment 7, wherein the yeast is capable of producing a test fermented green tea infusion with a pH of about 4 or less, less than about 7 g/L ethanol, less than about 0.5 g/L acetic acid, and less than about 5 g/L total sugar in a fermentation screening assay, the fermentation screening assay comprising preparing a test green tea infusion by contacting 10 g of green tea leaf with water at 80 °C for 45 minutes; adding 20 g/L sucrose or glucose to 5 ml of the test green tea infusion; adding 0.2 - 0.4 g/L of the yeast to the test green tea infusion; allowing the test green tea infusion to ferment at 28 °C for 6 days to produce the test fermented green tea infusion.

9. A fermented infusion according to any one of embodiments 6 to 8, wherein the fermentation microorganism added to the infusion does not comprise Gluconacetobacter xylinus, optionally wherein the fermentation microorganism added to the infusion does not comprise an acetic acid bacteria. 10. A process for preparing a fermented infusion of a plant material comprising a tea leaf, the process comprising the steps of:

(a) preparing an infusion of the plant material;

(b) adding carbohydrate to the infusion, optionally wherein the carbohydrate is a sugar;;

(c) adding a fermentation microorganism to the infusion; and

(d) fermenting the infusion under conditions suitable to the microorganism, to produce the fermented infusion, wherein at the end of fermentation step (d), the fermented infusion comprises: an acetic acid content of about 1 % (w/w) or less; an ethanol content of about 1.2% (v/v) or less; and a carbohydrate content of about 60 g/L or less, and/or a Brix value of about 6 ° Brix or less.

11. A process according to embodiment 10, wherein the infusion is fermented using a yeast, optionally wherein the yeast is a Saccharomyces cerevisiae yeast.

12. A process according to embodiment 10 or embodiment 11 , wherein the infusion is not fermented using a Gluconacetobacter xylinus bacteria, optionally wherein the infusion is not fermented using an acetic acid bacteria.

13. A process according to any one of embodiments 10 to 12, wherein the infusion is fermented for between 1 and 5 days, and/or wherein the infusion is fermented until the pH of the fermented infusion is less than about pH 4.

14. A process according to any one of embodiments 10 to 13, wherein the plant material comprises a green tea leaf.

15. A process according to any one of embodiments 10 to 14, wherein preparing the infusion in step (a) comprises contacting the plant material with water at a temperature of between about 60 °C and about 90 °C, optionally wherein the plant material is in contact with the water for between about 10 minutes and about 60 minutes prior to the fermentation microorganism being added.

16. A process according to any one of embodiments 10 to 15, wherein the total amount of carbohydrate added at the start of fermentation step (d) is from 30 - 50 g/L, and/or wherein the Brix value of the infusion at the start of fermentation step (d) is 3-5 ° Brix.

17. A process according to any one of embodiments 10 to 16, wherein the infusion is fermented until the carbohydrate content in the infusion is from 20 - 25 g/L, and/or wherein the infusion is fermented until the Brix value of the fermented infusion is 2-2.5 ° Brix. 18. A fermented infusion obtainable by the process of any one of embodiments 10 to 18.

19. A process according to any one of embodiments 10 to 17, wherein the fermented infusion is prepared as a beverage, optionally wherein the preparing the fermented infusion as a beverage comprises adding one or more additional ingredients to the fermented infusion, wherein the one or more additional ingredients are selected from: a flavouring agent, a stabiliser, a preservative, a sugar, a sweetener, a fruit juice, a colouring agent, a vitamin, a mineral, and an amino acid.

20. A beverage comprising a fermented infusion according to any one of embodiments 1 to 9 and 18, optionally wherein the beverage further comprises one or more additional ingredients selected from: a flavouring agent, a stabiliser, a preservative, a sugar, a sweetener, a fruit juice, a colouring agent, a vitamin, a mineral, and an amino acid.

21 . A beverage according to embodiment 20, wherein the beverage has an acetic acid content of about 1 % (w/w) or less, and an ethanol content of about 1.2% (v/v) or less.

22. A beverage according to embodiment 20 or embodiment 21 , wherein the beverage has a carbohydrate content of between about 20 g/L and about 60 g/L, and/or a Brix value of between about 2 ° Brix and 6 ° Brix; or wherein the beverage comprises a sweetener in an amount providing a sweetness in the beverage equivalent to between about 2 ° Brix and 6 ° Brix.

23. A fermented infusion of a plant material comprising a tea leaf, wherein the fermented infusion has:

- an acetic acid content of 1 .5% (w/w) or less;

- an ethanol content of about 1 .2% (v/v) or less; and

- a carbohydrate content of about 60 g/L or less, and/or a Brix value of about 6 ⁰ Brix or less.

24. A process for preparing a fermented infusion of a plant material comprising a tea leaf, the process comprising the steps of:

(a) providing an infusion of the plant material;

(b) adding carbohydrate to the infusion, optionally wherein the carbohydrate is a sugar;;

(c) adding a fermentation microorganism to the infusion; and

(d) fermenting the infusion under conditions suitable to the microorganism, to produce the fermented infusion, wherein at the end of fermentation step (d), the fermented infusion comprises: an acetic acid content of about 1% (w/w) or less; an ethanol content of about 1 .2% (v/v) or less; and a carbohydrate content of about 60 g/L or less, and/or a Brix value of about 6 ° Brix or less.