Fermented Ready-to-Drink Hemp Beverages

Abstract

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

Description

[Technical field]

[0001] The present invention relates to a ready-to-drink beverage composition comprising fermented hemp or a fermented hemp extract. A further aspect of the invention is a method for preparing the ready-to-drink beverage composition. [Background technology]

[0002] Hemp may be legally consumed in many jurisdictions in a variety of forms. Legal consumption of hemp, for example by brewing an infusion from hemp leaves and flowers, is becoming increasingly popular not only for medicinal uses but also as an enjoyable beverage with recognized health benefits. However, hemp infusions have a strong earthy and herbaceous taste that is not popular with consumers in general. Many people desire new flavors in their beverages, but do not like to consume flavorings, especially beverages with added synthetic flavors or sugary fruit juice drinks. Thus, there is commercial value in providing hemp beverages with novel flavors and without strong earthy / hemp notes.

[0003] Any reference to a prior art document in this specification should not be considered as an admission that such prior art is well known or forms part of the common general understanding in the art. As used herein, the words "comprises," "comprising," and similar words should not be construed in an exclusive or exhaustive sense. In other words, they are intended to mean "including, but not limited to." Summary of the Invention

[0004] The object of the present invention is to improve the state of the art and to provide a novel product which overcomes at least to some extent the above-mentioned inconveniences, or at least to provide a useful alternative. This object is achieved by the subject matter of the independent claims. The dependent claims further develop the idea of ​​the present invention.

[0005] Accordingly, in a first aspect, the present invention provides a beverage composition comprising fermented hemp or a fermented hemp extract, the beverage composition having a weight ratio of the sum of 3-methylbutyl acetate and 3-methylbutanol to 3-methylbutanal of greater than 2000, the beverage composition being a ready-to-drink beverage.

[0006] In a second aspect, the present invention provides a method for preparing a beverage composition, the method comprising: a. steeping hemp in water to produce an aqueous hemp extract and steeped hemp; b. fermenting the aqueous hemp extract in the presence or absence of steeped hemp; Fermentation is carried out by a yeast, including Pichia yeast, and the beverage composition is a ready-to-drink beverage.

[0007] A further aspect of the present invention provides the use of hemp fermented with Pichia kluyveri NYSC5485 (CNCM I-5525) for the production of a ready-to-drink beverage.

[0008] Hemp leaves can be made into beverages by steeping the dried leaves in either hot or cold water. These beverages have characteristic flavors such as hay, woody, earthy, aniseed, spicy and citrus notes. The inventors have surprisingly found that through fermentation with Pichia yeast, the ethanol content of the hemp beverage remains low, the stewed herb, earthy and spicy notes are reduced and an enhanced refreshing fruity flavor is developed. The piney, medicinal and grassy flavors characteristic of hemp, which some consumers find unpalatable, are reduced by fermentation with Pichia yeast, especially by fermentation with Pichia kluyveri NYSC5485. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0009] Thus, the present invention relates in part to a beverage composition comprising (e.g., consisting of) fermented hemp or fermented hemp extract having a weight ratio of the sum of 3-methylbutyl acetate and 3-methylbutanol to 3-methylbutanal greater than 2000, such as greater than 4000, such as greater than 8000, such as greater than 10000, such as greater than 12000, such as greater than 14000, or even such as greater than 16000. The aldehyde 3-methylbutanal contributes a malty, solvent-like note. The beverage composition may be a ready-to-drink beverage.

[0010] The ratio can be calculated from the weight concentrations in ppm as follows:

number

[0011] During fermentation of sugars by Pichia, higher esters such as 3-methylbutyl acetate are produced initially, but as fermentation progresses, 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 in the fermented beverage with reduced malty, beer, and solvent notes and masking the hemp notes.

[0012] In the context of the present invention, the term hemp refers to the flowers, seeds and leaves of the plant Cannabis sativa. Hemp may be, for example, chemotype II, III, IV or V of Cannabis sativa. It is to be understood that in the present invention, the term hemp does not refer to chemotype I of Cannabis sativa, commonly called cannabis. Hemp is a plant that contains low levels of the psychoactive component Δ 9 -tetrahydrocannabinol (THC). For example, hemp according to the invention may contain less than 0.2% THC on a dry matter basis. Hemp contains cannabidiol (CBD), which has been associated with therapeutic effects but does not have psychoactive effects. Hemp is also a valuable source of polyphenols such as gallic acid, chlorogenic acid, coumaric acid, quercetin and ellagic acid.

[0013] The beverage compositions of the present invention may include 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 compositions of the present invention may include cannabidiol in addition to those provided by the fermented hemp or fermented hemp extract.

[0014] In the context of the present invention, the term "fermentation" refers to a process in which microbial activity brings about a change (typically a desired change) in a food or beverage. Fermentation can be by yeast and / or bacteria. Fermentation can be anaerobic or aerobic. Fermentation is one of the oldest techniques used for food preservation and enrichment.

[0015] The term "fermented hemp" refers to hemp that has been subjected to a process in which the activity of microorganisms, such as yeast, results in chemical changes, typically desirable changes, in the organic components of the hemp. Fermented hemp may be fermented, for example, by yeast, in an aqueous medium. Fermented hemp may be dried hemp that has been added to an aqueous fermentation medium, fermented, and then separated from the aqueous fermentation medium and dried. Fermented hemp may also be fermented in a "solid-state" fermentation, for example, by adding a yeast starter culture to hemp leaves, e.g., fresh hemp leaves.

[0016] The term "hemp extract" refers to a material extracted from hemp, e.g., an aqueous extract of hemp. The hemp extract may be, for example, an aqueous extract from hemp leaves and stems after removal of oil from the leaves and stems. The term "fermented hemp extract" refers to a hemp extract after the hemp extract has been fermented, e.g., an aqueous hemp extract after the hemp extract has been fermented, such as by yeast. The fermented hemp extract may be a dried fermented hemp extract.

[0017] In one embodiment, the beverage composition (e.g., beverage) has a weight ratio of the sum of 2-methylbutyl acetate and 2-methylbutanol to 2-methylbutanal greater than 2000, e.g., greater than 3500. The aldehyde 2-methylbutanal contributes a malty, solvent-like note. The ratio can be calculated from the weight concentrations in ppm as follows:

number

[0018] During fermentation of sugars by Pichia, higher esters such as 2-methylbutyl acetate are produced initially, but as fermentation progresses, 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 pleasing aroma balance in which the malty, beer, and solvent notes in the fermented beverage are reduced, masking the hemp notes with these aroma markers.

[0019] In one embodiment, the beverage composition (e.g., 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, such as greater than 250, such as greater than 300, such as greater than 350, or even such as greater than 400. This ratio can be calculated from the weight concentrations in ppm as follows:

number

[0020] The compound 2,3-butanedione is also known as diacetyl. 2,3-butanedione 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 may be perceived as off-flavors in fermentation.

[0021] In one embodiment, the beverage composition comprises less than 10 g / kg acetic acid on a dry basis, such as less than 1 g / kg acetic acid on a dry basis, further such as less than 0.5 g / kg acetic acid on a dry basis. The acid "bite" in the flavor profile of beverages such as kombucha or kefir is not always desired by consumers seeking a milder flavor profile.

[0022] Many consumers are looking for complex, refreshing beverages as an alternative to alcoholic beverages. In one embodiment, the beverage composition has an ethanol content of less than 1.2% by weight, such as less than 0.5% by weight, such as less than 0.2% by weight, further such as less than 0.05% by weight.

[0023] The ready-to-drink beverage may be a fermented hemp infusion or a water flavored with fermented hemp. The ready-to-drink beverage may comprise 2-30% by weight hemp, for example 4-15% by weight hemp. The ready-to-drink beverage may further comprise other ingredients such as flavourings or stabilizers. The ready-to-drink beverage may be a carbonated beverage. The ready-to-drink beverage may further comprise a 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 further comprise an herb such as mint, verbena or basil. The ready-to-drink beverage may further comprise a flower extract such as hibiscus or rose. The ready-to-drink beverage may further comprise vegetable juice or vegetable extract, such as tomato, cucumber, kale and celery. The ready-to-drink beverage may further comprise a botanical infusion. The ready-to-drink beverage may further comprise dietary fibre, such as inulin, wheat dextrin, resistant starch and beta-glucan (e.g. from oat or barley). The ready-to-drink beverage may comprise added vitamins and minerals. The ready-to-drink beverage may comprise replicating or non-replicating beneficial microorganisms. The beneficial microorganisms may be, for example, 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 present invention may comprise a total of 10 to 100 mg cannabidiol per litre, for example 20 to 60 mg cannabidiol per litre.

[0024] 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 further comprise other ingredients, such as flavourings or stabilizers. The beverage liquid concentrate may comprise 4-60% by weight hemp, for example 8-30% by weight hemp. The beverage liquid concentrate may further comprise a 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 further comprise an herb, such as mint, verbena or basil. The beverage liquid concentrate may further comprise a flower extract, such as hibiscus or rose. The beverage liquid concentrate may further comprise a vegetable juice or vegetable extract, such as tomato, cucumber, kale and celery. The beverage liquid concentrate may further comprise dietary fibres such as inulin, wheat dextrin, resistant starch and beta-glucan (e.g. from oat or barley). The beverage liquid concentrate may further comprise botanical infusions. The beverage liquid concentrate may comprise added vitamins and minerals. The beverage liquid concentrate may comprise replicating or non-replicating beneficial microorganisms. The beneficial microorganisms may be, for example, probiotics.

[0025] One aspect of the invention provides a method for preparing a beverage composition, the method comprising: a. steeping hemp in water to produce an aqueous hemp extract and steeped hemp; b. fermenting the aqueous hemp extract in the presence or absence of steeped hemp; Fermentation is carried out by a yeast, including Pichia yeast, and the beverage composition is a ready-to-drink beverage.

[0026] The yeast may be predominantly Pichia yeast, e.g., greater than 50% of the microbial colony forming units present during the fermentation may be Pichia. For example, greater than 50% of the yeast colony forming units may be Pichia, e.g., greater than 60%, 70%, 80%, 90% of the yeast colony forming units may be Pichia. For example, essentially all yeast colony forming units present during the fermentation may be Pichia, and as a further example, essentially all microbial colony forming units present during the fermentation may be Pichia.

[0027] The beverage composition prepared by the method of the present 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 present invention may have an ethanol content of less than 1.2% by weight, such as less than 0.5% by weight, for example less than 0.2% by weight, further for example less than 0.05% by weight. In one embodiment, the beverage composition prepared by the method of the present invention is a beverage composition of the present invention.

[0028] The term steeping refers to steeping the material in a liquid. The hemp may be steeped in water for at least 2 minutes, such as at least 3 minutes, such as at least 4 minutes, such as at least 5 minutes, such as at least 10 minutes, such as at least 15 minutes, such as at least 20 minutes, such as at least 30 minutes, such as at least 60 minutes, and further such as at least 120 minutes. The components of the hemp are extracted into the water during the steeping process. The steeping may be carried out at a temperature between 4°C and 98°C, such as between 20°C and 95°C, such as between 60°C and 95°C. Hemp contains some fermentable sugars, but further fermentable sugars, such as sucrose or glucose, may be added to the hemp aqueous extract. The fermentable sugars may be fructose or glucose. The fermentable sugars may be in the form of honey. For example, fermentable sugars may be added to the hemp aqueous extract at a level of 2-10% by weight, such as 3-7% by weight. The hemp may be dried and / or milled prior to steeping.

[0029] If fermentation is performed in the absence of steeped hemp, the hemp can be steeped in water, for example using a continuous countercurrent extraction system. A further option is to steep the hemp in water using a batch process, and the steeped hemp is separated from the hemp aqueous extract using a filtration system. The hemp to water ratio on a dry basis during steeping can be 1:1 to 1:100 by weight, for example 1:2 to 1:95 by weight, for example 1:10 to 1:90 by weight, for example 1:30 to 1:70 by weight, further for example 1.3 to 1:6.

[0030] After steeping, the temperature of the aqueous hemp extract is adjusted to a temperature suitable for yeast growth, such as 20°C to 37°C, such as 25°C to 35°C, such as 28°C to 32°C. The temperature can be adjusted, for example, using a laminar flow heat exchanger. The yeast is added to the aqueous hemp extract to initiate fermentation. The fermentation of the aqueous hemp extract may be carried out at a temperature of 25°C to 35°C, such as 28°C to 32°C. The fermentation of the aqueous hemp extract may be carried out for at least 2 hours, such as at least 6 hours, such as at least 12 hours, such as at least 24 hours, further such as at least 48 hours.

[0031] The fermentation may be carried out at a pH of from 3 to 8, for example from 4 to 6.

[0032] The aqueous hemp extract may be fermented in the presence of other ingredients such as flavorings or stabilizers. The aqueous hemp extract may be fermented in the presence of other ingredients such as fruits or fruit juices, for example strawberries, citrus fruits (e.g. orange, grapefruit, lemon, lime, calamansi, bergamot, clementine, tangerine or yuzu), peaches, pomegranates, watermelon, blackberries, raspberries, persimmons or cranberries. The aqueous hemp extract may be fermented in the presence of other ingredients such as herbs, for example mint, verbena or basil. The aqueous hemp extract may be fermented in the presence of other ingredients such as flower extracts, for example hibiscus or rose. The aqueous hemp extract may be fermented in the presence of other ingredients such as vegetable juices or vegetable extracts, for example tomato, cucumber, kale or celery. The aqueous hemp extract may be fermented in the presence of other ingredients such as plant infusions. The aqueous hemp extract may be fermented in the presence of other ingredients such as added vitamins and minerals.

[0033] Fermentation may be carried out in the presence of a nitrogen source, such as yeast extract or yeast peptone. Fermentation may be carried out in the presence of amino acids, such as valine, leucine, isoleucine and / or phenylalanine, added, for example, at a level of 0.05% to 0.1% by weight of the aqueous hemp extract.

[0034] The fermentation may be carried out under anaerobic, microaerobic or aerobic conditions. For example, the oxygen partial pressure in the fermenter may be 0-30%, such as 1-20%, further such as 2-10%.

[0035] Preferably, the hemp has not been subjected to any chemical treatment, such as hydrolysis, prior to fermentation.

[0036] Fermentation should be continued for at least 10 3 CFU / g, e.g. at least 10 4 CFU / g, e.g. at least 10 5 CFU / g, e.g. at least 10 6 CFU / g, further e.g. at least 10 7This can be done by adding one or more yeasts at an initial mixed yeast level of CFU / g.

[0037] Fermentation may be carried out in the absence of acetic bacteria to avoid the development of an acid "hot" flavor profile, such as that found in kombucha or kefir. In one embodiment, fermentation is carried out in the absence of lactic acid bacteria.

[0038] In one embodiment, fermentation is performed in the absence of Saccharomyces subsp. For example, fermentation can be performed in the absence of Saccharomyces cerevisiae. S. cerevisiae is used in winemaking and beer brewing. In addition to producing alcohol, which may be undesirable, S. cerevisiae produces "fermented," wine-like, yeasty flavor notes rather than fresh, fruity notes.

[0039] In one embodiment, the fermentation is carried out in the absence of yeasts selected from the group consisting of Zygosaccharomyces bailii, Schizosaccharomyces pombe, Torulaspora delbreukii, Rhodotorula mucilaginosa, Brettanomyces bruxellensis and Candida stellate, which are commonly present in kombucha "tea-fungal" cultures.

[0040] The inventors have found that fermentation of hemp with Pichia kluyveri gives particularly good results. Beverage compositions containing hemp or hemp extract fermented with Pichia kluyveri have a pleasant fruity flavor with less smoky hemp notes than unfermented beverages. Pichia kluyveri occurs naturally throughout the world, for example in olives, grapes and coffee. In one embodiment, the Pichia yeast is predominantly Pichia kluyveri, for example, more than 50% of the microbial colony forming units present during the 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 of the yeast colony forming units present during the fermentation may be Pichia kluyveri, and as a further example, essentially all of the microbial colony forming units present during the fermentation may be Pichia kluyveri.

[0041] Pichia kluyveri NCYC246 (alternatively designated CBS188) is the type strain of Pichia kluyveri. Type strains are publicly available, for example from the National Collection of Yeast Cultures, Quadram Institute Bioscience (Norwich, UK).

[0042] In one embodiment, the Pichia yeast is Pichia kluyveri NCYC246. For example, greater than 50% of the yeast colony forming units present during the fermentation can be Pichia kluyveri NCYC246. For example, greater than 50% of the microbial colony forming units present during the fermentation can be Pichia kluyveri NCYC246. For example, essentially all of the yeast colony forming units present during the fermentation can be Pichia kluyveri NCYC246. As a further example, essentially all of the microbial colony forming units present during the fermentation can be Pichia kluyveri NCYC246.

[0043] Pichia kluyveri NCYC2781 (alternatively designated Maize Silage Isolate 86) is publicly available from, for example, the National Collection of Yeast Cultures, Quadram Institute Bioscience (Norwich, UK).

[0044] In one embodiment, the Pichia yeast is Pichia kluyveri NCYC2781. For example, greater than 50% of the yeast colony forming units present during the fermentation can be Pichia kluyveri NCYC2781. For example, greater than 50% of the microbial colony forming units present during the fermentation can be Pichia kluyveri NCYC2781. For example, essentially all of the yeast colony forming units present during the fermentation can be Pichia kluyveri NCYC2781. As a further example, essentially all of the microbial colony forming units present during the fermentation can be Pichia kluyveri NCYC2781.

[0045] Pichia kluyveri is widely distributed in nature and is present in fruits such as olives, grapes and coffee. Pichia kluyveri NYSC5485, collected and isolated from a coffee fermentation plant in Nicaragua, was deposited on June 23, 2020 at the Collection Nationale de Cultures de Microorganismes (CNCM), Institut Pasteur, 25rue du Docteur Roux, F-75724 PARIS Cedex15, France, and received the accession number CNCM I-5525.

[0046] In one embodiment, the yeast is Pichia kluyveri NYSC5485 (CNCM I-5525). For example, more than 50% of the yeast colony forming units present during the fermentation can be Pichia kluyveri NYSC5485 (CNCM I-5525). For example, more than 50% of the microbial colony forming units present during the fermentation can be Pichia kluyveri NYSC5485 (CNCM I-5525). For example, essentially all of the yeast colony forming units present during the fermentation can be Pichia kluyveri NYSC5485 (CNCM I-5525). As a further example, essentially all of the microbial colony forming units present during the fermentation can be Pichia kluyveri NYSC5485 (CNCM I-5525).

[0047] The inventors have found that particularly good results are obtained by fermenting hemp with Pichia kluyveri NYSC 5485. Fermenting hemp with Pichia kluyveri NYSC 5485 results in a higher weight ratio of the sum of 3-methylbutyl acetate and 3-methylbutanol to 3-methylbutanal, which corresponds to an improved aroma balance.

[0048] Fermentation should be continued for at least 10 3 CFU / g, e.g. at least 10 4 CFU / g, e.g. at least 10 5 CFU / g, e.g. at least 10 6 CFU / g, further e.g. at least 10 7 This can be achieved by adding Pichia kluyveri, for example Pichia kluyveri 5485 (CNCM I-5525), at an initial yeast level of CFU / g.

[0049] The gene sequence of Pichia kluyveri NYSC5485 (CNCM I-5525) was analyzed using PacBio sequencing technology. DNA purification was performed using Qiagen Gentra Puregene Yeast kit. 12 DNA fragments were identified. These fragments could be individual chromosomes, i.e. 11 chromosomes and one mitochondrial DNA (SEQ ID NO: 4). The gene sequences are submitted in electronic format as SEQ ID NO: 1 to SEQ ID NO: 12.

[0050] In one embodiment of the method of the present invention, the Pichia yeast comprises a gene sequence having at least 98% identity (e.g., at least 99% identity) to SEQ ID NO:1, a gene sequence having at least 98% identity (e.g., at least 99% identity) to SEQ ID NO:2, a gene sequence having at least 98% identity (e.g., at least 99% identity) to SEQ ID NO:3, a gene sequence having at least 98% identity (e.g., at least 99% identity) to SEQ ID NO:5, a gene sequence having at least 98% identity (e.g., at least 99% identity) to SEQ ID NO:6, These include a gene sequence having at least 99% identity to SEQ ID NO:7, a gene sequence having at least 98% identity (e.g. at least 99% identity) to SEQ ID NO:8, a gene sequence having at least 98% identity (e.g. at least 99% identity) to SEQ ID NO:9, a gene sequence having at least 99% identity to SEQ ID NO:10, a gene sequence having at least 98% identity (e.g. at least 99% identity) to SEQ ID NO:11, or a gene sequence having at least 98% identity (e.g. at least 99% identity) to SEQ ID NO:12.

[0051] In one embodiment of the method of the present invention, the Pichia yeast comprises a gene sequence having at least 98% identity (e.g., at least 99% identity) to SEQ ID NO:1, a gene sequence having at least 98% identity (e.g., at least 99% identity) to SEQ ID NO:2, a gene sequence having at least 98% identity (e.g., at least 99% identity) to SEQ ID NO:3, a gene sequence having at least 99.5% identity to SEQ ID NO:4, a gene sequence having at least 98% identity (e.g., at least 99% identity) to SEQ ID NO:5, a gene sequence having at least 98% identity (e.g., at least 99% identity) to SEQ ID NO:6, 9% identity) to SEQ ID NO:7, a gene sequence having at least 99% identity (e.g., at least 99% identity) to SEQ ID NO:8, a gene sequence having at least 98% identity (e.g., at least 99% identity) to SEQ ID NO:9, a gene sequence having at least 99% identity to SEQ ID NO:10, a gene sequence having at least 98% identity (e.g., at least 99% identity) to SEQ ID NO:11, and a gene sequence having at least 98% identity (e.g., at least 99% identity) to SEQ ID NO:12.

[0052] In one embodiment, the Pichia yeast comprises a genetic sequence having at least 98% (e.g., at least 99%) global nucleotide identity to the combination of SEQ ID NOs: 1-12. The term "combination of SEQ ID NOs: 1-12" means that a sequence identity comparison is made for all individual DNA fragments as if they were concatenated into a single sequence.

[0053] In one embodiment, the Pichia yeast comprises a gene sequence having at least 98% (e.g., at least 99%) identity to SEQ ID NO:1. In one embodiment, the Pichia yeast comprises a gene sequence having at least 98% (e.g., at least 99%) identity to SEQ ID NO:2. In one embodiment, the Pichia yeast comprises a gene sequence having at least 98% (e.g., at least 99%) identity to SEQ ID NO:3. In one embodiment, the Pichia yeast comprises a gene sequence having at least 98% (e.g., at least 99%) identity to SEQ ID NO:5. In one embodiment, the Pichia yeast comprises a gene sequence having at least 98% (e.g., at least 99%) identity to SEQ ID NO:6. In one embodiment, the Pichia yeast comprises a gene sequence having at least 98% (e.g., at least 99%) identity to SEQ ID NO:7. In one embodiment, the Pichia yeast comprises a gene sequence having at least 98% (e.g., at least 99%) identity to SEQ ID NO:8. In one embodiment, the Pichia yeast comprises a gene sequence having at least 98% (e.g., at least 99%) identity to SEQ ID NO:9. In one embodiment, the Pichia yeast comprises a gene sequence having at least 99% identity to SEQ ID NO: 10. In one embodiment, the Pichia yeast comprises a gene sequence having at least 98% (e.g., at least 99%) identity to SEQ ID NO: 11. In one embodiment, the Pichia yeast comprises a gene sequence having at least 98% (e.g., at least 99%) identity to SEQ ID NO: 12.

[0054] Names referring to yeasts may change over time, and it should be understood that a yeast according to the methods of the invention may contain the genetic sequence identities described in the preceding paragraph for the Pichia yeasts of the invention, regardless of the name applied to the yeast.

[0055] One aspect of the invention provides a method of preparing a beverage composition, the method comprising: a) steeping hemp in water to produce an aqueous hemp extract and steeped hemp; b) fermenting the aqueous hemp extract in the presence or absence of steeped hemp, wherein the fermentation is carried out with a yeast comprising a gene sequence having at least 98% (e.g., at least 99%) overall nucleotide identity to a combination of SEQ ID NOs: 1-12.

[0056] In one embodiment, the aqueous hemp extract is separated from the steeped hemp after fermentation. For example, the steeped hemp can be separated from the aqueous hemp extract using a filtration system. After fermentation, the aqueous hemp extract becomes a fermented hemp extract.

[0057] The fermented hemp extract may be packaged as a ready-to-drink beverage, for example packaged in a bottle or can. The fermented hemp extract may have a concentration of 2-60% total dissolved solids by weight, for example, 2-25% total dissolved solids by weight. The fermented hemp extract may be packaged as a ready-to-drink beverage after adding additional ingredients such as flavorings and preservatives. The fermented hemp extract may be heat treated to inactivate or kill yeast and any spoilage organisms prior to packaging. The fermented hemp extract may be passed through a filter to remove yeast and spoilage organisms prior to packaging.

[0058] The fermented hemp extract may be combined with other ingredients such as fruits or juices, herbal extracts, flower extracts, vegetable juices or extracts, botanical infusions, vitamins and minerals, and beneficial microorganisms. Cannabidiol may be added to the fermented hemp extract, for example, the cannabidiol may be emulsified in an aqueous solution of the fermented hemp extract using an emulsifier.

[0059] In one embodiment, the aqueous hemp extract may be concentrated after fermentation, for example as an intermediate product for the production of a ready-to-drink product at a different location. The fermented hemp extract may have a total dissolved solids concentration of 25-60% by weight, further for example, a total dissolved solids concentration of 50-60% by weight. The dissolved solids may also be measured, for example, by passing the extract through a fine filter to remove undissolved solids, weighing the filtrate to determine the total weight, evaporating the filtrate, and then weighing the residue to obtain the weight of the dissolved solids. Additional ingredients, such as flavorings and preservatives, may be added to the liquid concentrate.

[0060] The fermented aqueous hemp extract (e.g., fermented hemp extract) may be concentrated to produce a powder, such as a soluble beverage powder. For example, the fermented aqueous hemp extract (fermented hemp extract) may be dried by spray drying or freeze drying to produce a soluble beverage powder. For example, the fermented aqueous hemp extract may be spray dried with a carrier, such as maltodextrin, to produce a soluble beverage powder.

[0061] The post-fermentation aqueous hemp extract may be spray dried along with other ingredients such as fruit or juice, herbal extracts, flower extracts, vegetable juice or extracts, plant infusions, vitamins and minerals, and beneficial microorganisms.

[0062] The soluble beverage powders may be used, for example, as intermediate products for the production of ready-to-drink products at a different location. The soluble beverage powders may be combined with other raw materials, for example dry raw materials such as tea, herbal teas, dried fruit, soluble coffee or roasted and ground coffee. The soluble beverage powders may be combined with other raw materials, such as fruit powders, herbs, vegetable powders, vitamins, minerals, and beneficial microorganisms.

[0063] One aspect of the present invention provides the use of hemp fermented with Pichia kluyveri NYSC5485 (CNCM I-5525) for the production of a ready-to-drink beverage.

[0064] The fermented hemp according to the invention can be obtained in "solid state" fermentation, for example by adding a starter culture of Pichia yeast to hemp leaves, for example fresh, undried hemp leaves. The term "solid state" fermentation refers to fermentation carried out in solid material. In solid state fermentation, liquid water is present, but this term is used in contrast to liquid fermentation, such as may occur in an aqueous solution in a fermentation vessel.

[0065] One aspect of the invention provides a method for preparing a beverage composition, the method comprising: adding a yeast starter culture to the hemp leaves, the starter culture having at least 10 yeast cells per gram of hemp leaves; 4 CFU (e.g., at least 10 5 CFU, further e.g. at least 10 6 providing yeast in a concentration of 100 CFU; A process of fermenting hemp leaves; drying the fermented hemp leaves to produce fermented hemp; extracting the fermented hemp with water to produce a ready-to-drink beverage; Includes.

[0066] The yeast starter culture may be added to the hemp leaves as a liquid or powder. The hemp leaves may have a moisture content of 45-55% by weight when the yeast starter culture is added. The leaves may be steam treated prior to fermentation to reduce the amount of environmental microorganisms before adding the yeast. Fermentation may occur at a solids content of more than 40% by weight, such as more than 45% by weight, further such as more than 50% by weight.

[0067] While fermentation continues for some time, drying of the fermented hemp may be initiated. For example, the hemp leaves may be placed in a container such as a tank, a starter culture may be added, and then fermentation may proceed for 1-4 days, after which the fermented hemp may be removed from the container and dried. As a further example, after the starter culture is added to the hemp leaves, the hemp leaves may be gradually dried, for example, on racks or hanging trays. Once fermentation has begun, the hemp leaves may be gradually dried as fermentation continues. For example, the fermented hemp leaves may be dried to a moisture content of less than 20% by weight (e.g., less than 15% by weight) over a period of 1-30 days, such as 4-20 days. The drying temperature may be less than about 40° C., for example, about 25° C. to 35° C. A fan may 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.

[0068] The yeast may be a Pichia, such as Pichia kluyveri, further such as Pichia kluyveri NYSC5485.

[0069] The ready-to-drink beverage may be filled into bottles or cans. The fermented hemp may be combined with other extractable ingredients such as tea, herbal teas, dried fruit, soluble coffee or roasted and ground coffee before being extracted with water to produce the ready-to-drink beverage.

[0070] Those skilled in the art will understand that all features of the invention disclosed herein can be freely combined. In particular, features described for the product of the invention can be combined with the method of the invention, and vice versa. Furthermore, features described for different embodiments of the invention can be combined. Where known equivalents exist for specific features, such equivalents are incorporated herein as if specifically mentioned.

[0071] Further advantages and features of the invention are apparent from the drawings and non-limiting examples. EXAMPLES

[0072] Example 1: Hemp fermentation method Organic hemp leaves (Coop Naturaplan, Switzerland) were used as starting material to prepare the infusion. First, a hot infusion was made by adding 20 g of dry starting material in 1000 mL of water at 100° C. and steeping for 10 minutes. The infusion was then placed in an ice bath until the temperature dropped below 30° C. The infusion was then filtered through a sieve to further remove small pieces and / or leaves. The filtrate (hemp aqueous extract) was distributed in equal amounts as a control (Ref) or to the various fermentation samples.

[0073] Fermented hemp was produced by fermenting the infusion with glucose (2% m / m) and various starter cultures including Pichia kluyveri NYSC5485 (CNCM I-5525), P. kluyveri NCYC246 (reference strain of Pichia kluyveri), P. kluyveri NCYC2781, commercial brewer's yeast Saccharomyces cerevisiae (SafAle S-33, LeSaffre, France), and symbiotic culture of bacteria and yeasts (SCOBY) pellicles from kombucha tea fermentation. All strains (except SCOBY) were grown in edible yeast-peptone liquid medium from glycerol vials and inoculated into the reference hemp brew at an optical density (OD600nm) of 0.5. All inoculated infusions were stored at 30°C under microaerobic conditions for 24 h. After incubation, each fermentation exudate was filter sterilized (0.2 μm).

[0074] Example 2: Sensory analysis of hemp fermented with different yeasts The sensory profiles of different variations of hemp infusions were tasted. The control was hemp taste, which had smoky, herbaceous, woody, and earthy notes. The hemp infusion fermented with Saccharomyces cerevisiae had strong malty and sour notes along with off-notes. The hemp fermented with SCOBY had a very strong sour taste. The hemp infusions fermented with two different Pichia yeasts were fruity and had a significantly reduced hemp / smoky note compared to the control. No buttery, sour, or fatty off-notes were identified in the hemp fermented with Pichia yeast. The hemp fermented with Pichia kluyveri NYSC5485 could be considered to have a more balanced aroma profile than the hemp fermented with Pichia kluyveri NCYC246.

[0075] Example 3: Analysis of flavor profiles focusing on aroma compounds present in hemp fermented with different yeasts Quantitative profiling of volatile compounds in hemp infusions was performed by headspace / solid phase microextraction coupled with gas chromatography and mass spectrometry (HS / SPME-GC-MS) using a gas chromatograph (Agilent Technology 8890 GC System) coupled to a mass spectrometer (Agilent Technology 7010B TQ).

[0076] First, 1.0 mL of hemp infusion samples were incubated in 10 mL screw-top headspace vials at 30°C for 10 min, and then extracted using SPME fibers (PDMS / DVB, Supelco) at 30°C for 10 min. All vials were placed in a tray cooled to 6°C prior to analysis. Volatiles were thermally desorbed from the SPME fibers in splitless mode at 250°C and separated on a capillary column (DB-WAX, Agilent). The gas chromatograph oven temperature was programmed to be initially at 35°C for 5 min, then increased to 230°C at 4°C / min, and then at 230°C for 10 min. Helium was used as the carrier gas at a flow rate of 1 mL / min. Target compounds were identified by pure standards and quantified with MSD ChemStation software (Agilent). All samples were run in duplicate.

[0077] The levels in ppm 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 after 24 hours are shown in the following table. [Table 1]

[0078] The SCOBY fermented sample had high levels of acetic acid equivalent to 190 g / kg on a dry basis.

[0079] The weight ratios A, B, and C of the aroma components are shown in the table below.

number

[0080] Samples fermented with Pichia yeasts (NYSC5485, NCYC2781 and NCYC246) show higher Ratio A, Ratio B and Ratio C than the control and samples fermented with S. cerevisiae or SCOBY. P. kluyveri NYSC5485 gives higher values ​​for Ratio A, Ratio B and Ratio C than the reference strain P. kluyveri NCYC246 and higher values ​​for Ratio A and Ratio B than P. kluyveri NCYC2781.

[0081] The levels of terpenes and terpenoids present in the samples were measured. The following target compounds were identified by NIST library and retention index. The peak area of ​​each compound was integrated over the entire sample and the ratio of fermented to non-fermented samples is shown below. All of these compounds have been reported and detected in hemp extracts or hemp oil [VRLJ Bloemendal et al., Org. Biomol. Chem., 18, 3203-3215 (2020). DOI 10.1039 / D0OB00464B] [ASWanas 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].

[0082] All samples were measured in duplicate. [Table 3]

[0083] Terpenes and terpenoids are major contributors to the "hempiness" characteristic, which is perceived as a combination of piney, medicinal and grassy flavor notes. After fermentation with Pichia kluyveri yeast, the hempiness was significantly reduced. P. kluyveri NYSC5485 was found to reduce these compounds associated with hempiness characteristics to a greater extent than the reference strain P. kluyveri NYSC246.

[0084] Example 4: Investigation of the effect of aroma ratio on sensory perception by spikes Solutions were prepared with different levels of 3-methylbutyl acetate, 3-methylbutanol and 3-methylbutanal added. In one set of solutions, the compounds were added to water and in the other set, the compounds were added to an unfermented hemp extract control (Ref).

[0085] The samples were tasted by a six-person expert taste panel and comments were collected. The samples and comments are shown below. [Table 4] [Table 5]

[0086] It is important to have the right balance between esters / alcohols and aldehydes. It has been observed that high levels of esters and low levels of aldehydes in hemp infusions, for example ratios A above 2000, can mask the "hemp-like" notes.

[0087] Example 5: Comparison of the gene sequence of P. kluyveri NYSC5485 with that of the reference strain P. kluyveri NCYC246 The gene sequences of Pichia kluyveri NYSC5485 (CNCM I-5525) and P. kluyveri NCYC246 were analyzed using PacBio sequencing technology. DNA purification was performed using the Qiagen Gentra Puregene Yeast kit. 12 DNA fragments were identified for P. kluyveri NYSC5485 and 15 DNA fragments were identified for the type strain P. kluyveri NCYC246. The gene sequence of P. kluyveri NYSC5485 was submitted in electronic form as SEQ ID NO:1 to SEQ ID NO:12, and for P. kluyveri NCYC246 as SEQ ID NO:13 to SEQ ID NO:27. Mitochondrial DNA is SEQ ID NO:4 for P. kluyveri NYSC5485 and SEQ ID NO:20 for P. kluyveri NCYC246.

[0088] The gene sequence of Pichia kluyveri NYSC5485 (CNCM I-5525) was compared with P. kluyveri NCYC246 using the software OrthoANIu. The overall average nucleotide identity was found to be 97.91%.

[0089] The correspondence between the various sequences is shown in the table below. The multiple sequences of Pichia kluyveri NYSC5485 correspond most closely to the combination of sequences of P. kluyveri NCYC246 and vice versa. [Table 6]

[0090] The type strain P. kluyveri NCYC246 contained the gene sequences with the greatest identity to each of SEQ ID NOs: 1-12 as shown below: 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 NO:10, 98.33%; SEQ ID NO:11, 97.526%; SEQ ID NO:12, 97.77%.

Claims

1. 1. A beverage composition comprising fermented hemp or a 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 wherein the beverage composition is a ready-to-drink beverage.

2. 2. The beverage composition of claim 1, wherein the weight ratio of the sum of 2-methylbutyl acetate and 2-methylbutanol to 2-methylbutanal is greater than 2,000.

3. 3. The beverage composition according to claim 1, wherein the weight ratio of the sum of 3-methylbutyl acetate, 2-methylbutyl acetate, isobutyl acetate, 2-phenylethyl acetate, and 2-phenylethanol to 2,3-butanedione is greater than 50.

4. 3. A beverage composition according to claim 1 or 2, having an ethanol content of less than 1.2% by weight.

5. 1. A method for preparing a beverage composition, comprising: a. steeping hemp in water to produce an aqueous hemp extract and steeped hemp; b. fermenting the aqueous hemp extract in the presence or absence of the steeped hemp; The method, wherein the fermentation is carried out with a yeast comprising Pichia yeast, and the beverage composition is a ready-to-drink beverage.

6. 6. The method of claim 5, wherein the Pichia yeast is Pichia kluyveri.

7. The method of claim 5 or 6, wherein the Pichia yeast comprises a gene sequence having at least 98% overall nucleotide identity to a combination of SEQ ID NOs: 1 to 12.

8. 7. The method of claim 5 or 6, wherein the fermentation is carried out in the absence of Saccharomyces cerevisiae.

9. 7. The method according to claim 5 or 6, wherein the fermentation is carried out in the absence of acetic acid bacteria.

10. 7. The method of claim 5 or 6, comprising separating the aqueous hemp extract from steeped hemp after fermentation.

11. Use of hemp fermented with Pichia kluyveri NYSC5485 (CNCM I-5525) for producing a ready-to-drink beverage.