Field of the Invention

The present invention relates to a ready to drink beverage composition comprising fermented hemp or fermented hemp extract. A further aspect of the invention is a method of preparing a ready to drink beverage composition.

Background of the Invention

Hemp may legally be consumed in various forms in a number of jurisdictions. Consumption of legal hemp, for example by brewing an infusion from leaves and flowers of hemp is increasingly popular not just for medicinal use, but also as an enjoyable beverage with perceived health benefits. However, hemp infusion has a strong earthy and herbaceous taste that is not popular with all consumers. Many people desire new flavours in their beverages, but would prefer not to consume beverages with added flavouring, especially synthetic flavourings or sugary fruit juices. The provision of hemp beverages with novel flavours and a less strong earthy/hemp note would therefore be of commercial value.

Any reference to prior art documents in this specification is not to be considered an admission that such prior art is widely known or forms part of the common general knowledge in the field. As used in this specification, the words "comprises", "comprising", and similar words, are not to be interpreted in an exclusive or exhaustive sense. In other words, they are intended to mean "including, but not limited to".

Summary of the invention

An object of the present invention is to improve the state of the art and to provide a new product to overcome at least some of the inconveniences described above, or at least to provide a useful alternative. The object of the present invention is achieved by the subject matter of the independent claims. The dependent claims further develop the idea of the present invention.

Accordingly, the present invention provides in a first aspect a beverage composition comprising fermented hemp or fermented hemp extract wherein the weight ratio of the sum of 3-methylbutyl acetate and 3-methylbutanol to 3-methylbutanal is greater than 2000 and the beverage composition is a ready to drink beverage.

In a second aspect, the invention provides a method of preparing a beverage composition comprising; a. steeping hemp in water to form an aqueous hemp extract and steeped hemp; and b. fermenting the aqueous hemp extract in the presence or absence of the steeped hemp; wherein the fermentation is performed by yeast comprising Pichia yeast and the beverage composition is a ready to drink beverage.

A further aspect of the invention provides use of hemp fermented with Pichia kluyveri NYSC 5485 (CNCM 1-5525) to produce a ready to drink beverage.

Hemp leaves can be made into beverages by steeping the dried leaves in either hot or cold water. These beverages have a characteristic flavour, including hay, woody, earthy, aniseed, spicy and citrus notes. The inventors were surprised to find that, through fermentation with Pichia yeast, the hemp beverage remained low in ethanol, but developed an enhanced refreshing fruity flavour with the stewed herb, earthy and spicy notes being reduced. The piney, medicinal and grassy flavour characteristic of hemp, which some consumers find unpalatable, was reduced by fermentation with Pichia yeast, especially by fermentation with Pichia kluyveri NYSC 5485. Detailed Description of the invention

Consequently the present invention relates in part to a beverage composition comprising (for example consisting of) fermented hemp or fermented hemp extract wherein the weight ratio of the sum of 3-methylbutyl acetate and 3-methylbutanol to 3-methylbutanal is greater than 2000, for example greater than 4000, for example greater than 8000, for example greater than 10000, for example greater than 12000, for example greater than 14000, for further example greater than 16000. The aldehyde 3-methylbutanal contributes a malty, solvent-like note. The beverage composition may be a ready to drink beverage.

The ratio can be calculated from the weight concentrations in parts per million as follows:

Higher esters such as 3-methylbutyl acetate are initially produced during the fermentation of sugars with Pichia, but as the fermentation proceeds, higher alcohols such as 3-methylbutanol are produced. A high ratio of the sum of 3-methylbutyl acetate and 3-methylbutanol to 3-methylbutanal corresponds to a pleasant aroma balance with reduced malty, beer and solvent notes in the fermented beverage, masking the hemp note.

In the context of the present invention, the term hemp refers to the flower, seeds and leaves of the plant Cannabis sativa. The hemp may for example be Cannabis sativa chemotype II, III, IV or V. It should be understood that the term hemp in the present invention does not refer to Cannabis sativa chemotype I, which is commonly referred to as cannabis. Hemp has low levels of the psychoactive component A ⁹ -tetrahydrocannabinol (THC). For example, the hemp according to the present invention may contain less than 0.2% THC on a dry matter basis. Hemp comprises cannabidiol (CBD) which has been linked with therapeutic effects but does not have psychoactive effects. Hemp is also a valuable source of polyphenols such as gallic acid, chlorogenic acid, cumaric acid, quercetin and ellagic acid.

The beverage composition of the invention may comprise cannabinoids in addition to those provided by the fermented hemp or fermented hemp extract. The cannabinoids may be isomers and/or derivatives of cannabinoids. The cannabinoid derivatives may be cannabinoid acids. The beverage composition of the invention may comprise cannabidiol in addition to that provided by the fermented hemp or fermented hemp extract.

In the context of the present invention the term "fermentation" refers to a process in which the activity of microorganisms brings about a change (typically a desirable change) to a foodstuff or beverage. The fermentation may be with yeasts and/or bacteria. The fermentation may be anaerobic or aerobic. Fermentation is one of the oldest means of preserving and enhancing foods.

The term "fermented hemp" refers to hemp which has been subjected to a process in which the activity of microorganisms such as yeasts brings about a chemical change in the organic components of the hemp, typically a desirable change. The fermented hemp may have been fermented in an aqueous medium, for example with yeast. The fermented hemp may be a dried hemp which had been added to an aqueous fermentation medium and fermented before being separated from the aqueous fermentation medium and dried. The fermented hemp may have been fermented in a "solid-state" fermentation, for example adding a starter culture of yeast to hemp leaves, for example fresh hemp leaves.

The term "hemp extract" refers to material extracted from hemp, for example an aqueous extract of hemp. The hemp extract may for example be an aqueous extract from hemp leaves and stems following oil removal from the leaves and stems. The term "fermented hemp extract" refers to an extract of hemp where the hemp extract has been fermented, for example an aqueous hemp extract where the aqueous extract has been fermented, such as by yeast. The fermented hemp extract may be a dried fermented hemp extract.

In an embodiment the beverage composition (for example the beverage) has a weight ratio of the sum of 2-methylbutyl acetate and 2-methylbutanol to 2-methylbutanal is greater than 2000, for example greater than 3500. The aldehyde 2-methylbutanal and contributes a malty, solvent-like note. The ratio can be calculated from the weight concentrations in parts per million as follows:

Higher esters such as 2-methylbutyl acetate are initially produced during the fermentation of sugars with Pichia, but as the fermentation proceeds, higher alcohols such as 2-methylbutanol are produced. A high ratio of the sum of 2-methylbutyl acetate and 2-methylbutanol to 2-methylbutanal corresponds to a pleasant aroma balance with reduced malty, beer and solvent notes in the fermented beverage, masking the hemp note with these aroma markers.

In an embodiment the beverage composition (for example the beverage) has a weight ratio of the sum of 3-methylbutyl acetate, 2-methylbutyl acetate, isobutyl acetate, 2- phenylethyl acetate and 2-phenylethanol to 2,3-butanedione greater than 50, for example greater than 250, for example greater than 300, for example greater than 350, for further example greater than 400. This ratio can be calculated from the weight concentrations in parts per million as follows:

The compound 2,3-butanedione is also known as diacetyl. It provides a buttery or fatty note. A high ratio of the sum of 3-methylbutyl acetate, 2-methylbutyl acetate, isobutyl acetate, 2-phenylethyl acetate and 2-phenylethanol to 2,3-butanedione corresponds to enhanced fruity and floral notes without the buttery and fatty notes that can be perceived as off flavour in a fermentation.

In an embodiment, the beverage composition comprises less than 10 g/kg acetic acid on a dry basis, for example less than 1 g/kg acetic acid on a dry basis, for further example less than 0.5 g/kg acetic acid on a dry basis. The acid "bite" in the flavour profile of beverages such as kombucha or kefir is not always desired by consumers who seek a gentler flavour profile.

Many consumers seek beverages with a complex, refreshing taste as a replacement to alcoholic drinks. In an embodiment, the beverage composition has an ethanol content below 1.2wt%, for example below 0.5wt%, for example below 0.2wt%, for further example below 0.05wt%.

The ready to drink beverage may be a fermented hemp infusion or a water flavoured with fermented hemp. The ready to drink beverage may comprise between 2 and 30 wt.% hemp, for example between 4 and 15 wt.% hemp. The ready to drink beverage may additionally comprise other components such as flavourings or stabilizers. The ready to drink beverage may be carbonated. The ready to drink beverage may additionally comprise fruit or fruit juice, such as strawberry, citrus fruit (e.g. orange, grapefruit, lemon, lime, calamansi, bergamot, clementine, tangerine or yuzu), peach, pomegranate, watermelon, blackberry, raspberry, persimmon or cranberry. The ready to drink beverage may additionally comprise herbs such as mint, verbena or basil. The ready to drink beverage may additionally comprise flower extracts such as hibiscus or rose. The ready to drink beverage may additionally comprise vegetable juices or vegetable extracts such as tomato, cucumber, kale and celery. The ready to drink beverage may additionally comprise botanical infusions. The ready to drink beverage may additionally comprise dietary fibre such as inulin, wheat dextrin, resistant starches and beta-glucans, e.g. from oats or barley. The ready to drink beverage may comprise added vitamins and minerals. The ready to drink beverage may comprise beneficial micro-organisms, replicating or non-replicating. The beneficial microorganisms may for example be probiotics. The ready to drink beverage may contain cannabidiol in addition to the cannabidiol provided by the fermented hemp or fermented hemp extract. The ready to drink beverage of the invention may comprise a total of between 10 and 100 mg cannabidiol per litre, for example between 20 and 60 mg cannabidiol per litre.

The ready to drink beverage may be prepared from a beverage liquid concentrate such as a concentrated fermented hemp infusion. The beverage liquid concentrate may additionally comprise other components such as flavourings or stabilizers. The beverage liquid concentrate may comprise between 4 and 60 wt.% hemp, for example between 8 and 30 wt.% hemp. The beverage liquid concentrate may additionally comprise fruit or fruit juice, such as strawberry, citrus fruit (e.g. orange, grapefruit, lemon, lime, calamansi, bergamot, clementine, tangerine or yuzu), peach, pomegranate, watermelon, blackberry, raspberry, persimmon or cranberry. The beverage liquid concentrate may additionally comprise herbs such as mint, verbena or basil. The beverage liquid concentrate may additionally comprise flower extracts such as hibiscus or rose. The beverage liquid concentrate may additionally comprise vegetable juices or vegetable extracts such as tomato, cucumber, kale and celery. The beverage liquid concentrate may additionally comprise dietary fibre such as inulin, wheat dextrin, resistant starches and beta-glucans, e.g. from oats or barley. The beverage liquid concentrate may additionally comprise botanical infusions. The beverage liquid concentrate may comprise added vitamins and minerals. The beverage liquid concentrate may comprise beneficial micro-organisms, replicating or non- replicating. The beneficial microorganisms may for example be probiotics.

An aspect of the invention provides a method of preparing a beverage composition comprising; a. steeping hemp in water to form an aqueous hemp extract and steeped hemp; and b. fermenting the aqueous hemp extract in the presence or absence of the steeped hemp; wherein the fermentation is performed by yeast comprising Pichia yeast and the beverage composition is a ready to drink beverage.

The yeast may be predominantly Pichia yeast, for example, more than 50% of the microorganism colony forming units present during fermentation may be Pichia. For example more than 50% of the yeast colony forming units may be Pichia, for example more than 60, 70, 80, 90% of the yeast colony forming units may be Pichia. For example, essentially all the yeast colony forming units present during fermentation may be Pichia, for further example, essentially all the micro-organism colony forming units present during fermentation may be Pichia.

The beverage composition prepared by the method of the invention may be a hemp beverage, for example a beverage comprising fermented hemp or fermented hemp extract. The beverage composition prepared by the method of the invention may have an ethanol content below 1.2wt%, for example below 0.5wt%, for example below 0.2wt%, for further example below 0.05wt%. In an embodiment, the beverage composition prepared by the method of the invention is the beverage composition of the invention.

The term steeping refers to immersing and soaking a material in liquid. The hemp may be steeped in water for at least 2 minutes, for example at least 3 minutes, for example at least 4 minutes, for example at least 5 minutes, for example at least 10 minutes, for example at least 15 minutes, for example at least 20 minutes, for example at least 30 minutes, for example at least 60 minutes, for further example at least 120 minutes. During the steeping process, components of the hemp are extracted into the water. The steeping may be performed at a temperature between 4°C and 98°C, for example between 20°C and 95°C, for example between 60°C and 95°C. Although hemp contains some fermentable sugar, further fermentable sugar may be added to the aqueous hemp extract, for example sucrose or glucose. The fermentable sugar may be fructose or glucose. The fermentable sugar may be in the form of honey. For example fermentable sugar may be added to the aqueous hemp extract at a level of between 2 and 10 wt.%, for example between 3 and 7 wt.%. The hemp may be dried and/or ground before steeping.

When fermentation is to be performed in the absence of the steeped hemp, the hemp may for example be steeped in water using a continuous counter-current extraction system. A further option is to steep the hemp in water using a batch process, with the steeped hemp being separated from the aqueous hemp extract using a filter system. The ratio of hemp on a dry basis to water during steeping may be between 1:1 and 1:100 by weight, for example between 1:2 and 1:95 by weight, for example between 1:10 and 1:90 by weight, for example between 1:30 and 1:70 by weight, for further example between 1.3 and 1:6.

After steeping, the temperature of the aqueous hemp extract is adjusted to a temperature suitable for yeast growth, for example between 20°C and 37°C, for example between 25°C and 35°C, for example between 28°C and 32°C. The temperature may for example be adjusted using a laminar flow heat exchanger. Yeast is added to the aqueous hemp extract to commence fermentation. Fermenting the aqueous hemp extract maybe performed at a temperature between 25°C and 35°C, for example between 28°C and 32°C. Fermenting the aqueous hemp extract may be performed for at least 2 hours, for example at least 6 hours, for example at least 12 hours, for example at least 24 hours, for further example at least 48 hours.

The fermentation may be performed at a pH between 3 and 8, for example between 4 and 6. The aqueous hemp extract may be fermented in the presence of other components such as flavourings or stabilizers. The aqueous hemp extract may be fermented in the presence of other components such as fruit or fruit juice, for example strawberry, citrus fruit (e.g. orange, grapefruit, lemon, lime, calamansi, bergamot, clementine, tangerine or yuzu), peach, pomegranate, watermelon, blackberry, raspberry, persimmon or cranberry. The aqueous hemp extract may be fermented in the presence of other components such as herbs, e.g. mint, verbena or basil. The aqueous hemp extract may be fermented in the presence of other components such as flower extracts e.g. hibiscus or rose. The aqueous hemp extract may be fermented in the presence of other components such as vegetable juices or vegetable extracts e.g. tomato, cucumber, kale or celery. The aqueous hemp extract may be fermented in the presence of other components such as botanical infusions. The aqueous hemp extract may be fermented in the presence of other components such as added vitamins and minerals.

The fermentation may be performed in the presence of a nitrogen source such as yeast extract or yeast peptone. The fermentation may be performed in the presence of added amino acids such as valine, leucine, isoleucine and/or phenylalanine, for example at a level of between 0.05wt.% and 0.1wt.% of the aqueous hemp extract.

The fermentation may be performed in anaerobic, microaerobic or aerobic conditions. For example, the partial oxygen pressure in the fermenter may be from 0 to 30%, for example from 1 to 20%, for further example from 2 to 10%.

Preferably the hemp has not been subjected to chemical processing such as hydrolysis before fermentation.

The fermentation may be performed by adding one or more yeasts at an initial combined yeast level of at least 10 ³ CFU/g, for example at least 10 ⁴ CFU/g, for example at least 10 ⁵ CFU/g, for example at least 10 ⁶ CFU/g, for further example at least 10 ⁷ CFU/g. The fermentation may be performed in the absence of acetic acid bacteria to avoid generating an acid "bite" in the flavour profile such as is found in kombucha or kefir. In an embodiment the fermentation is performed in the absence of lactic acid bacteria.

In an embodiment the fermentation is performed in the absence of Saccharomyces subspecies. For example the fermentation may be performed in the absence of Saccharomyces cerevisiae. S. cerevisiae is used for winemaking and beer brewing. In addition to generating alcohol which may not be desired, S. cerevisiae generates a "fermented", winey, yeasty flavour note rather than a fresh fruity note.

In an embodiment the fermentation is performed in the absence of a yeast selected from the group consisting of Zygosaccharomyces bailii, Schizosaccharomyces pombe, Torulaspora delbreuckii, Rhodotorula mucilaginosa, Brettanomyces bruxellensis and Candida stellate. These yeasts are commonly found in kombucha "tea-fungus" cultures.

The inventors found that fermentation of hemp with Pichia kluyveri gave particularly good results. A beverage composition comprising hemp or hemp extract fermented with Pichia kluyveri had a pleasant fruity flavour with less smoky hemp note than the unfermented beverage. Pichia kluyveri occurs naturally around the world, for example on olives, grapes and coffee. In an embodiment the Pichia yeast is predominantly Pichia kluyveri, for example, more than 50% of the microorganism colony forming units present during fermentation may be Pichia kluyveri. For example more than 50% of the yeast colony forming units may be Pichia kluyveri, for example more than 60, 70, 80, 90% of the yeast colony forming units may be Pichia kluyveri. For example, essentially all the yeast colony forming units present during fermentation may be Pichia kluyveri, for further example, essentially all the micro-organism colony forming units present during fermentation may be Pichia kluyveri. Pichia kluyveri NCYC 246 (alternatively designated CBS 188) is the type-strain of Pichia kluyveri. It is publicly available, for example from the National Collection of Yeast Cultures, Quadram Institute Bioscience, Norwich, UK.

In an embodiment the Pichia yeast is Pichia kluyveri NCYC 246. For example more than 50% of the yeast colony forming units present during fermentation may be Pichia kluyveri NCYC 246. For example, more than 50% of the microorganism colony forming units present during fermentation may be Pichia kluyveri NCYC 246. For example, essentially all the yeast colony forming units present during fermentation may be Pichia kluyveri NCYC 246. For further example, essentially all the microorganism colony forming units present during fermentation may be Pichia kluyveri NCYC 246.

Pichia kluyveri NCYC 2781 (alternatively designated Maize Silage Isolate 86) is publicly available, for example from the National Collection of Yeast Cultures, Quadram Institute Bioscience, Norwich, UK.

In an embodiment the Pichia yeast is Pichia kluyveri NCYC 2781. For example more than 50% of the yeast colony forming units present during fermentation may be Pichia kluyveri NCYC 2781. For example, more than 50% of the microorganism colony forming units present during fermentation may be Pichia kluyveri NCYC 2781. For example, essentially all the yeast colony forming units present during fermentation may be Pichia kluyveri NCYC 2781. For further example, essentially all the microorganism colony forming units present during fermentation may be Pichia kluyveri NCYC 2781.

Pichia kluyveri is widely distributed in nature, for example being found on fruits such as olives, grapes and coffee. Pichia kluyveri NYSC 5485, collected and isolated from a coffee fermentation in Nicaragua, was deposited with the Collection Nationale de Cultures de Microorganismes (CNCM), Institut Pasteur, 25 rue du Docteur Roux, F- 75724 PARIS Cedex 15, France, on 23 ^rd June 2020 and given the deposit number CNCM 1-5525. In an embodiment, the yeast is Pichia kluyveri NYSC 5485 (CNCM 1-5525). For example more than 50% of the yeast colony forming units present during fermentation may be Pichia kluyveri NYSC 5485 (CNCM 1-5525). For example, more than 50% of the microorganism colony forming units present during fermentation may be Pichia kluyveri NYSC 5485 (CNCM 1-5525). For example, essentially all the yeast colony forming units present during fermentation may be Pichia kluyveri NYSC 5485 (CNCM I- 5525), For further example, essentially all the microorganism colony forming units present during fermentation may be Pichia kluyveri NYSC 5485 (CNCM 1-5525).

The inventors have found that especially good results were obtained by fermenting hemp with Pichia kluyveri NYSC 5485. Fermenting hemp with Pichia kluyveri NYSC 5485 resulted in a higher weight ratio of the sum of 3-methylbutyl acetate and 3- methylbutanol to 3-methylbutanal, corresponding to an enhanced aroma balance.

The fermentation may be performed by adding Pichia kluyveri, for example Pichia kluyveri NYSC 5485 (CNCM 1-5525), at an initial yeast level of at least 10 ³ CFU/g, for example at least 10 ⁴ CFU/g, for example at least 10 ⁵ CFU/g, for example at least 10 ⁶ CFU/g, for further example at least 10 ⁷ CFU/g.

The genetic sequence of Pichia kluyveri NYSC 5485 (CNCM 1-5525) was analysed using PacBio sequencing technology. DNA purification was performed using a Qiagen Gentra Puregene Yeast kit. Twelve DNA fragments were identified. These may be individual chromosomes; 11 chromosomes and 1 mitochondrion DNA (SEQ ID NO:4). The genetic sequences are filed in electronic form as SEQ ID NO:1 - SEQ ID NO:12.

In an embodiment of the method of the invention, the Pichia yeast comprises a genetic sequence having at least 98% (for example at least 99% identity) to SEQ ID NO:1, a genetic sequence having at least 98% identity (for example at least 99% identity) to SEQ ID NO:2, a genetic sequence having at least 98% identity (for example at least 99% identity) to SEQ ID NO:3, a genetic sequence having at least 98% identity (for example at least 99% identity) to SEQ ID NO:5, a genetic sequence having at least 98% identity (for example at least 99% identity) to SEQ ID NO:6, a genetic sequence having at least 99% identity to SEQ ID NO:7, a genetic sequence having at least 98% identity (for example at least 99% identity) to SEQ ID NO:8, a genetic sequence having at least 98% identity (for example at least 99% identity) to SEQ ID NO:9, a genetic sequence having at least 99% identity to SEQ ID NQ:10, a genetic sequence having at least 98% identity (for example at least 99% identity) to SEQ ID NO:11, or a genetic sequence having at least 98% identity (for example at least 99% identity) to SEQ ID NO:12.

In an embodiment of the method of the invention, the Pichia yeast comprises a genetic sequence having at least 98% (for example at least 99% identity) to SEQ ID NO:1, a genetic sequence having at least 98% identity (for example at least 99% identity) to SEQ ID NO:2, a genetic sequence having at least 98% identity (for example at least 99% identity) to SEQ ID NO:3, a genetic sequence having at least 99.5% identity to SEQ ID NO:4, a genetic sequence having at least 98% identity (for example at least 99% identity) to SEQ ID NO:5, a genetic sequence having at least 98% identity (for example at least 99% identity) to SEQ ID NO:6, a genetic sequence having at least 99% identity to SEQ ID NO:7, a genetic sequence having at least 98% identity (for example at least 99% identity) to SEQ ID NO:8, a genetic sequence having at least 98% identity (for example at least 99% identity) to SEQ ID NO:9, a genetic sequence having at least 99% identity to SEQ ID NQ:10, a genetic sequence having at least 98% identity (for example at least 99% identity) to SEQ ID NO:11 and a genetic sequence having at least 98% identity (for example at least 99% identity) to SEQ ID NO:12.

In an embodiment, the Pichia yeast comprises a genetic sequence having a global nucleotide identity at least 98% (for example at least 99%) to the combination of SEQ ID NOs:1-12. By the term "combination of SEQ ID NOs:1-12" it is meant that the sequence identity comparison is performed against all the individual DNA fragments as if concatenated into a single sequence. In an embodiment the Pichia yeast comprises a genetic sequence having at least 98% (for example at least 99%) identity to SEQ ID NO:1. In an embodiment the Pichia yeast comprises a genetic sequence having at least 98% (for example at least 99%) identity to SEQ ID NO:2. In an embodiment the Pichia yeast comprises a genetic sequence having at least 98% (for example at least 99%) identity to SEQ ID NO:3. In an embodiment the Pichia yeast comprises a genetic sequence having at least 98% (for example at least 99%) identity to SEQ ID NO:5. In an embodiment the Pichia yeast comprises a genetic sequence having at least 98% (for example at least 99%) identity to SEQ ID NO:6. In an embodiment the Pichia yeast comprises a genetic sequence having at least 99% identity to SEQ ID NO:7. In an embodiment the Pichia yeast comprises a genetic sequence having at least 98% (for example at least 99%) identity to SEQ ID NO:8. In an embodiment the Pichia yeast comprises a genetic sequence having at least 98% (for example at least 99%) identity to SEQ ID NO:9. In an embodiment the Pichia yeast comprises a genetic sequence having at least 99% identity to SEQ ID NQ:10. In an embodiment the Pichia yeast comprises a genetic sequence having at least 98% (for example at least 99%) identity to SEQ ID NO:11. In an embodiment the Pichia yeast comprises a genetic sequence having at least 98% (for example at least 99%) identity to SEQ ID NO:12.

The names by which yeasts are referred to may change with time. It should be understood that the yeast according to the method of the invention may comprise the genetic sequence identities described in the preceding paragraphs for the Pichia yeast of the invention regardless of the name applied to the yeast.

An aspect of the invention provides a method of preparing a beverage composition comprising; a) steeping hemp in water to form an aqueous hemp extract and steeped hemp; and b) fermenting the aqueous hemp extract in the presence or absence of the steeped hemp; wherein the fermentation is performed by a yeast comprising a genetic sequence having a global nucleotide identity at least 98% (for example at least 99%) to the combination of SEQ ID NOs:1-12. In an embodiment, the aqueous hemp extract is separated from the steeped hemp after fermentation. For example the steeped hemp may be separated from the aqueous hemp extract using a filter system. After fermentation, the aqueous hemp extract becomes fermented hemp extract.

The fermented hemp extract may be packaged as a ready-to-drink beverage, for example packaged in a bottle or a can. The fermented hemp extract may have a concentration of between 2 and 60 wt.% total dissolved solids, for example a concentration of 2 to 25 wt.% total dissolved solids. Additional ingredients such as flavours and preservatives may be added to the fermented hemp extract before it is packaged as a ready-to-drink beverage. The fermented hemp extract may be heat- treated to de-activate or kill the yeast and any spoilage organisms before being packaged. The fermented hemp extract may be passed through a filter to remove the yeast and any spoilage organisms before being packaged.

The fermented hemp extract may be combined with other ingredients such as fruit or fruit juice, extracts of herbs, flower extracts, vegetable juices or vegetable extracts, botanical infusions, vitamins and minerals and beneficial micro-organisms. Cannabidiol may be added to the fermented hemp extract, for example cannabidiol may be emulsified into an aqueous solution of the fermented hemp extract with the aid of an emulsifier.

In an embodiment the aqueous hemp extract is concentrated after being fermented, for example as an intermediate product for the manufacture of ready-to-drink products, for example in a different location. The fermented hemp extract may have a concentration of 25 to 60 wt.% total dissolved solids, for further example a concentration of 50 to 60 wt.% total dissolved solids. The dissolved solids may for example be measured by passing the extract through a fine filter to remove non- dissolved solids, weighing the filtrate to establish the total weight, evaporating the filtrate and then weighing the residue to obtain the weight of the dissolved solids. Additional ingredients such as flavours and preservatives may be added to the liquid concentrate.

The aqueous hemp extract after fermentation (for example the fermented hemp extract) may be concentrated to form a powder, such as a soluble beverage powder. For example, the aqueous hemp extract after fermentation (the fermented hemp extract) may be dried by spray drying or freeze drying to form a soluble beverage powder. For example, the aqueous hemp extract after fermentation may be spray dried together with a carrier such as maltodextrin to form a soluble beverage powder.

The aqueous hemp extract after fermentation may be spray dried together with other ingredients such as fruit or fruit juice, extracts of herbs, flower extracts, vegetable juices or vegetable extracts, botanical infusions, vitamins and minerals and beneficial micro-organisms.

The soluble beverage powder may be used as an intermediate product for the manufacture of ready-to-drink products, for example in a different location. The soluble beverage powder may be combined with other ingredients, for example dry ingredients such as tea, herbal tea, dried fruit, soluble coffee or roast and ground coffee. The soluble beverage powder may be combined with other ingredients such as fruit powders, herbs, vegetable powders, vitamins, minerals and beneficial micro- organisms.

An aspect of the invention provides for the use of hemp fermented with Pichia kluyveri NYSC 5485 (CNCM 1-5525) to produce a ready to drink beverage.

Fermented hemp according to the invention may be obtained in a "solid-state" fermentation, for example adding a starter culture of Pichia yeast to hemp leaves, for example fresh hemp leaves which have not been dried. The term "solid-state" fermentation refers to fermentation taking place on solid materials. There is liquid water present in a solid-state fermentation, but the term is used in contrast to a liquid fermentation such as may occur in aqueous solution in a fermentation vessel.

An aspect of the invention provides a method of preparing a beverage composition comprising; adding a starter culture of yeast to hemp leaves, wherein the starter culture provides at least 10 ⁴ CFU (for example at least 10 ⁵ CFU, for further example at least 10 ⁶ CFU) of yeast per gram of hemp leaves; fermenting the hemp leaves; drying the fermented hemp leaves to form fermented hemp; and; extracting the fermented hemp with water to form a ready-to-drink beverage

The starter culture of yeast may be added as a liquid or a powder to the hemp leaves. The hemp leaves may have a moisture content of between 45 and 55 wt.% when the starter culture of yeast is added. The leaves may be treated with steam before fermentation to reduce the quantity of environmental micro-organisms before adding yeast. The fermentation may occur at a solids content greater than 40 wt.%, for example greater than 45 wt.%, for further example greater than 50 wt.%.

Drying the fermented hemp may commence while some fermentation continues. For example, the hemp leaves may be placed in a container such as a tank, the starter culture added and then fermentation allowed to proceed for 1-4 days before the fermented hemp is removed from the container and dried. For further example, the starter culture may be added to the hemp leaves before the hemp leaves are slowly dried, for example on a rack or suspended tray. Once fermentation has started, the hemp leaves may be slowly dried whilst fermentation continues. For example the fermented hemp leaves may be dried to a moisture content of below 20 wt.% (for example below 15 wt.%) over a period of between 1 and 30 days, for example between 4 and 20 days. The drying temperature may be less than about 40°C, for example between about 25°C and 35°C. A fan can be used to blow dry air over the material until the hemp leaves are dry. The fermented hemp leaves may be dried in the sun.

The yeast may be Pichia, for example Pichia kluyveri, for further example Pichia kluyveri NYSC 5485.

The ready-to-drink beverage may be filled into a bottle or can. The fermented hemp may be combined with other extractable materials such as tea, herbal tea, dried fruit, soluble coffee or roast and ground coffee before being extracted with water to form a ready-to-drink beverage.

Those skilled in the art will understand that they can freely combine all features of the present invention disclosed herein. In particular, features described for the product of the present invention may be combined with the method of the present invention and vice versa. Further, features described for different embodiments of the present invention may be combined. Where known equivalents exist to specific features, such equivalents are incorporated as if specifically referred to in this specification.

Further advantages and features of the present invention are apparent from the figures and non-limiting examples.

Examples

Example 1: Method of fermenting hemp

An infusion was prepared using organic hemp leaves (Coop Naturaplan, Switzerland) as starting material. A hot infusion was firstly made by adding 20 g of dry starting material in 1000 ml of water at 100°C, and steepingfor 10 min. Afterwards, the infusion was placed in an ice bath until the temperature fell below 30°C. The infusion was then filtered through sieves to remove additional pieces and/or leaves. The filtrate (aqueous hemp extract) was distributed equally as control (Ref) or for different fermentation variants. Fermented hemp was made by fermenting the infusion with glucose (2% m/m) and different starter cultures, including Pichia kluyveri NYSC 5485 (CNCM 1-5525), P. kluyveri NCYC 246 (the type strain of Pichia kluyveri), P. kluyveri NCYC 2781, commercial brewer's yeast Saccharomyces cerevisiae (SafAle S-33, LeSaffre, France) and a symbiotic culture of bacteria and yeasts (SCOBY) pellicle from Kombucha tea fermentation. All strains (except for SCOBY) were grown from glycerol vial in a food- grade yeast-peptone liquid medium, and inoculated in the reference hemp brew with optical density (OD at 600nm) of 0.5. All the inoculated infusions were stored at microaerobic condition at 30°C for 24 h. After the incubation, each fermented infusion underwent filter sterilization (0.2 μm).

Example 2: Sensory analysis of hemp fermented with different yeasts

The sensory profiles of different variations of hemp infusions were tasted. The control tasted of hemp, with smoky, herbaceous, woody, and earthy notes. The hemp infusion fermented with Saccharomyces cerevisiae had strong malty and acidic character with off notes. The hemp fermented with SCOBY had a very sharp acidity. The hemp infusions fermented with the two different Pichia yeasts were fruity, with a greatly reduced hemp/smoky note compared to the control. No buttery, sour, and fatty off notes were identified in the hemp fermented with Pichia yeasts. Hemp fermented with Pichia kluyveri NYSC 5485 could be considered to have a more balanced aroma profile to that fermented with Pichia kluyveri NCYC 246.

Example 3: Analysis of flavor profiles with a focus on aroma compounds present in hemp fermented with different yeasts.

Quantitative volatile profiling of the hemp infusions was conducted by headspace/solid phase microextraction coupled with gas chromatogram and mass spectrometry (HS/SPME-GC-MS). The apparatus used was a gas chromatograph (Agilent Technology 8890 GC System) coupled with a mass spectrometer (Agilent Technology 7010B TQ). Initially, 1.0 ml of the hemp infusion samples were incubated in a 10-mL screw-top headspace vial at 30°C for 10 min, followed by extraction using a SPME fiber (PDMS/DVB, Supelco) at 30°C for 10 min. All vials were placed in a tray cooled at 6°C before analysis. The volatiles were thermally desorbed from the SPME fiber at 250°C in splitless mode and resolved with a capillary column (DB-WAX, Agilent). The gas chromatograph oven temperature was programed initially at 35°C for 5 min, then raised to 230°C at 4°C/min, then at 230°C for 10 min. Helium was used as the carrier gas at a flow rate of 1 mL/min. The targeted compounds were identified by pure standards and quantified in MSD ChemStation software (Agilent). All samples were measured in duplicate.

Levels of 3-methylbutyl acetate, 3-methylbutanol, 3-methylbutanal, 2-methylbutyl acetate, 2-methylbutanol, 2-methylbutanal, isobutyl acetate, 2-phenylethyl acetate, 2- phenylethanol, 2,3-butanedione, ethanol and acetic acid in parts per million after 24 hours are listed in the table below.

The sample fermented with SCOBY had a high level of acetic acid, corresponding to 190 g/kg on a dry basis.

The weight ratios A, B and C of the aroma components are in the following table.

The samples fermented with a Pichia yeast (NYSC5485, NCYC 2781 and NCYC246) show higher ratios A, B and C than the control and the samples fermented with S. cerevisiae or SCOBY. P. kluyveri NYSC 5485 delivers a higher value for ratios A, B and C than the type strain P. kluyveri NCYC 246 and higher values for ratios A and B than P. kluyveri NCYC 2781.

The levels of terpenes and terpenoids present in the samples were measured. The targeted compounds below were identified through NIST library and retention index. Peak area of each compound was integrated across samples, and the ratio of fermented sample against non-fermented sample was shown below. All of these compounds have been reported and detected in hemp extraction or hemp oil, [V.R.L.J. Bloemendal et al., Org. Biomol. Chem., 18, 3203-3215 (2020). DOI 10.1039/D0OB00464B] [A.S. Wanas et al., "Chemical Composition of Volatile Oils of Fresh and Air-Dried Buds of Cannabis chemovars, Their Insecticidal and Repellent Activities", Natural Product Communications, May 19, 2020. DOI

10.1177/1934578X20926729],

All samples were measured in duplicate.

Terpenes and terpenoids are the main contributors to the "hempiness" character, which is perceived as a combination of piney, medicinal and grassy flavour notes. After fermentation with Pichia kluyveri yeasts the hempiness was significantly reduced. P. kluyveri NYSC 5485 was found to reduce these compounds associated with hempiness character more than the type strain P. kluyveri NCYC 246.

Example 4: Sensory impact of aroma ratio investigated by spiking

Solutions were made up with additions of 3-methylbutyl acetate, 3-methlybutanol and 3-methylbutanal at different levels. In one set of solutions the compounds were added to water, while in the other set, the compounds were added to the unfermented hemp extract control (Ref).

The samples were tasted by a technical taste panel of 6 persons and comments compiled. The samples and comments are below.

It is important to have the right balance between ester/alcohol and the aldehyde. It was observed that in hemp infusion, high levels of ester with low levels of aldehyde, for example Ratio A greater than 2000, was able to mask the "hempy" note.

Example 5: Comparison of genetic sequence of P. kluyveri NYSC 5485 to the type strain P. kluyveri NCYC 246.

The genetic sequence of Pichia kluyveri NYSC 5485 (CNCM 1-5525) and P. kluyveri NCYC

246 were analysed using PacBio sequencing technology. DNA purification was performed using a Qiagen Gentra Puregene Yeast kit. Twelve DNA fragments were identified for P. kluyveri NYSC 5485 and 15 for the type strain P. kluyveri NCYC 246. The genetic sequences for P. kluyveri NYSC 5485 are filed in electronic form as SEQ ID NO:1 - SEQ ID NO:12 and the genetic sequences for P. kluyveri NCYC 246 filed as SEQ ID NO:13 - SEQ ID NO:27. The mitochondrion DNA was SEQ ID NO:4 for P. kluyveri NYSC 5485 and SEQ ID NQ:20 for P. kluyveri NCYC 246. The genetic sequence of Pichia kluyveri NYSC 5485 (CNCM 1-5525) was compared with that of P. kluyveri NCYC 246 using the software OrthoANIu. The global average nucleotide identity was found to be 97.91%.

Correspondences between the different sequences are given in the table below. Some sequences for Pichia kluyveri NYSC 5485 correspond most closely with a combination of sequences for P. kluyveri NCYC 246 and vice versa.

The type strain P. kluyveri NCYC 246 comprised a genetic sequence with a maximum identity to each SEQ ID NO:1-12 taken individually as follows: SEQ ID NO:1, 97.85%; SEQ ID NO:2, 97.40%; SEQ ID NO:3, 97.86%; SEQ ID NO:4, 99.02%; SEQ ID NO:5, 98.05%; SEQ ID NO:6, 97.93%; SEQ ID NO:7, 98.12%; SEQ ID NO:8, 97.07%; SEQ ID NO:9, 97.20%; SEQ ID NQ:10, 98.33%; SEQ ID NO:!!, 97.526%; SEQ ID NO:12, 97.77%.